Compositions and methods comprising sequences having meganuclease activity

ABSTRACT

Compositions and methods comprising polynucleotides and polypeptides having meganuclease activity are provided. Further provided are nucleic acid constructs, yeast, plants, plant cells, explants, seeds and grain having the meganuclease sequences. Various methods of employing the meganuclease sequences are provided. Such methods include, for example, methods for producing a meganuclease with increased activity at a wide range of temperatures, methods for producing a yeast, plant, plant cell, explant or seed comprising a meganuclease with increased activity.

This application is a divisional of U.S. patent application Ser. No.13/886,317, filed May 3, 2013, now U.S. Pat. No. 9,499,827, issued Nov.22, 2016, which claims the benefit of U.S. Provisional Application No.61/642,470, filed May, 4, 2012 and U.S. Provisional Application No.61/683,765, filed Aug. 16, 2012; both of which are hereby incorporatedherein in their entirety by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named20161011_BB2117USDIV_SequenceLisiting.txt created on Oct. 11, 2016 andhaving a size of 929 kilobytes and is filed concurrently with thespecification. The sequence listing contained in this ASCII formatteddocument is part of the specification and is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

This invention is in the field of molecular biology. More specifically,this invention pertains to sequences having meganuclease activity.

BACKGROUND OF THE INVENTION

Recombinant DNA technology has made it possible to insert foreign DNAsequences into the genome of an organism, thus, altering the organism'sphenotype. The most commonly used plant transformation methods areAgrobacterium infection and biolistic particle bombardment in whichtransgenes integrate into a plant genome in a random fashion and in anunpredictable copy number. Thus, efforts are undertaken to controltransgene integration in plants.

Site-specific integration techniques, which employ site-specificrecombination systems, as well as, other types of recombinationtechnologies, have been used to generate targeted insertions of genes ofinterest in a variety of organism.

Other methods for inserting or modifying a DNA sequence involvehomologous DNA recombination by introducing a transgenic DNA sequenceflanked by sequences homologous to the genomic target. U.S. Pat. No.5,527,695 describes transforming eukaryotic cells with DNA sequencesthat are targeted to a predetermined sequence of the eukaryote's DNA.Transformed cells are identified through use of a selectable markerincluded as a part of the introduced DNA sequences.

While both systems have provided useful techniques for targetedinsertion of sequences of interest, there remains a need for nucleasesthat will facilitate precise modification of a plant or yeast genome. Inaddition, there remains a need for meganucleases with increased activitythat can introduce a double strand brake at a wide range oftemperatures.

BRIEF SUMMARY OF THE INVENTION

Compositions and methods comprising polynucleotides and polypeptideshaving meganuclease activity are provided. Further provided arecompositions comprising polynucleotides encoding variant meganucleasescomprising at least one amino acid modification, wherein the variantmeganuclease has increased activity. Also provided are nucleic acidconstructs, yeast, plants, plant cells, explants, seeds and grain havingthe meganuclease sequences.

Various methods of employing the meganuclease sequences are provided.Such methods include methods for increasing meganuclease activity in acell, yeast cell, plant plant cell, plant, explant or seed. Furtherprovided are methods and compositions that allow the variousmeganuclease polypeptides and variants and fragments thereof to beexpressed in a yeast or plant cell at a wide range of temperatures. Suchmethods and compositions find use in producing yeast, plant cells,plants and explants with improved meganuclease activity.

Thus in a first embodiment, the invention concerns an isolated orrecombinant polynucleotide comprising a nucleotide sequence encoding ameganuclease polypeptide, said polypeptide comprising: a) an amino acidsequence having at least one amino acid modification at an amino acidposition corresponding to a position of SEQ ID NO: 1 selected from thegroup consisting of positions 2, 12, 16, 22, 23, 31, 36, 43, 50, 56, 58,59, 62, 71, 72, 73, 80, 81, 82, 86, 91, 95, 98, 103, 113, 114, 116, 117,118, 121, 124, 128, 129, 131, 147, 151, 153, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,194, 195, 196, 197, 200, 203, 204, 209, 222, 232, 236, 237, 246, 254,258, 267, 278, 281, 282, 289, 308, 311, 312, 316, 318, 319, 334, 339,340, 342, 345, 346, 348 and combinations thereof; or, b) an amino acidsequence having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44 of any of the aminoacid modification of (a).

In other embodiments, the invention concerns an isolated or recombinantpolynucleotide of the present disclosure, wherein said nucleotidesequence encodes a meganuclease polypeptide having at least 80%, 81, %,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1.

In another embodiment, the invention concerns the isolated orrecombinant polynucleotide of embodiment 1, and its correspondingpolypeptide, wherein said at least one amino acid modificationcomprises; a) an aspartic acid (D) at a position corresponding to aminoacid position 2 in SEQ ID NO: 1; b) a histidine (H) at a positioncorresponding to amino acid position 12 in SEQ ID NO: 1; c) anisoleucine (I) at a position corresponding to amino acid position 16 inSEQ ID NO: 1; d) a cysteine (C) at a position corresponding to aminoacid position 22 in SEQ ID NO: 1; e) a leucine (L) at a positioncorresponding to amino acid position 23 in SEQ ID NO: 1; f) an arginine(R) at a position corresponding to amino acid position 31 in SEQ ID NO:1; g) an asparagine (N) at a position corresponding to amino acidposition 36 in SEQ ID NO: 1; h) a leucine (L) at a positioncorresponding to amino acid position 43 in SEQ ID NO: 1; i) an arginine(R) or lysine (K) at a position corresponding to amino acid position 50in SEQ ID NO: 1; j) a leucine (L) at a position corresponding to aminoacid position 56 in SEQ ID NO: 1; k) an isoleucine (I) at a positioncorresponding to amino acid position 58 in SEQ ID NO: 1; 1) a histidine(H) or alanine (A) at a position corresponding to amino acid position 59in SEQ ID NO: 1; m) a valine (V) at a position corresponding to aminoacid position 62 in SEQ ID NO: 1; n) a lysine (K) at a positioncorresponding to amino acid position 71 in SEQ ID NO: 1; o) a threonine(T) at a position corresponding to amino acid position 72 in SEQ ID NO:1; p) an alanine (A) at a position corresponding to amino acid position73 in SEQ ID NO: 1; q) an arginine (R) at a position corresponding toamino acid position 80 in SEQ ID NO: 1; r) a lysine (K) at a positioncorresponding to amino acid position 81 in SEQ ID NO: 1; s) an arginine(R) at a position corresponding to amino acid position 82 in SEQ ID NO:1; t) an aspartic acid (D) at a position corresponding to amino acidposition 86 in SEQ ID NO: 1; u) an isoleucine (I) at a positioncorresponding to amino acid position 91 in SEQ ID NO: 1; v) anisoleucine (I) at a position corresponding to amino acid position 95 inSEQ ID NO: 1; w) an arginine (R) at a position corresponding to aminoacid position 98 in SEQ ID NO: 1; x) a valine (V) at a positioncorresponding to amino acid position 103 in SEQ ID NO: 1; y) a serine(S) at a position corresponding to amino acid position 113 in SEQ ID NO:1; z) a proline (P) at a position corresponding to amino acid position114 in SEQ ID NO: 1; aa) an arginine (R) at a position corresponding toamino acid position 116 in SEQ ID NO: 1; bb) a glycine (G) at a positioncorresponding to amino acid position 117 in SEQ ID NO: 1; cc) athreonine (T) at a position corresponding to amino acid position 118 inSEQ ID NO: 1; dd) an glycine (G) at a position corresponding to aminoacid position 121 in SEQ ID NO: 1; ee) an arginine (R) at a positioncorresponding to amino acid position 124 in SEQ ID NO: 1; ff) a cysteine(C) at a position corresponding to amino acid position 128 in SEQ ID NO:1; gg) an alanine (A) at a position corresponding to amino acid position129 in SEQ ID NO: 1; hh) an arginine (R) at a position corresponding toamino acid position 131 in SEQ ID NO: 1; ii) a serine (S) at a positioncorresponding to amino acid position 147 in SEQ ID NO: 1; jj) an alanine(A) at a position corresponding to amino acid position 151 in SEQ ID NO:1; kk) a leucine (L) or a methionine (M) at a position corresponding toamino acid position 153 in SEQ ID NO: 1; ll) a tryptophan (W) at aposition corresponding to amino acid position 159 in SEQ ID NO: 1; mm) aglutamic acid (E) at a position corresponding to amino acid position 160in SEQ ID NO: 1; nn) a valine (V) at a position corresponding to aminoacid position 161 in SEQ ID NO: 1; oo) a tyrosine (Y) at a positioncorresponding to amino acid position 162 in SEQ ID NO: 1; pp) anarginine (R) at a position corresponding to amino acid position 163 inSEQ ID NO: 1; qq) a histidine (H) at a position corresponding to aminoacid position 164 in SEQ ID NO: 1; rr) a leucine (L) at a positioncorresponding to amino acid position 165 in SEQ ID NO: 1; ss) anarginine (R) at a position corresponding to amino acid position 166 inSEQ ID NO: 1; tt) a histidine (H) at a position corresponding to aminoacid position 167 in SEQ ID NO: 1; uu) a proline (P) at a positioncorresponding to amino acid position 168 in SEQ ID NO: 1; vv) an alanine(A) at a position corresponding to amino acid position 169 in SEQ ID NO:1; ww) a proline (P) at a position corresponding to amino acid position170 in SEQ ID NO: 1; xx) a histidine (H) at a position corresponding toamino acid position 171 in SEQ ID NO: 1; yy) a proline (P) at a positioncorresponding to amino acid position 172 in SEQ ID NO: 1; zz) anarginine (R) at a position corresponding to amino acid position 173 inSEQ ID NO: 1; aaa) a leucine (L) at a position corresponding to aminoacid position 174 in SEQ ID NO: 1; bbb) a proline (P) at a positioncorresponding to amino acid position 175 in SEQ ID NO: 1; ccc) aglutamine (Q) at a position corresponding to amino acid position 176 inSEQ ID NO: 1; ddd) an alanine (A) at a position corresponding to aminoacid position 177 in SEQ ID NO: 1; eee) an arginine (R) at a positioncorresponding to amino acid position 178 in SEQ ID NO: 1; fff) a valine(V) at a position corresponding to amino acid position 179 in SEQ ID NO:1; ggg) a glutamine (Q) at a position corresponding to amino acidposition 180 in SEQ ID NO: 1; hhh) a valine (V) at a positioncorresponding to amino acid position 182 in SEQ ID NO: 1; iii) a proline(P) at a position corresponding to amino acid position 183 in SEQ ID NO:1; jjj) a lysine (K) at a position corresponding to amino acid position184 in SEQ ID NO: 1; kkk) a threonine (T) or a histidine (H) at aposition corresponding to amino acid position 185 in SEQ ID NO: 1; lll)a serine (S) at a position corresponding to amino acid position 186 inSEQ ID NO: 1; mmm) a glutamic acid (E) at a position corresponding toamino acid position 187 in SEQ ID NO: 1; nnn) a leucine (L) at aposition corresponding to amino acid position 188 in SEQ ID NO: 1; ooo)a glutamic acid (E) at a position corresponding to amino acid position189 in SEQ ID NO: 1; ppp) a glutamine (Q) at a position corresponding toamino acid position 190 in SEQ ID NO: 1; qqq) a leucine (L) at aposition corresponding to amino acid position 191 in SEQ ID NO: 1; rrr)a proline (P) at a position corresponding to amino acid position 194 inSEQ ID NO: 1; sss) a lysine (K) at a position corresponding to aminoacid position 195 in SEQ ID NO: 1; ttt) a serine (S) at a positioncorresponding to amino acid position 196 in SEQ ID NO: 1; uuu) aphenylalanine (F) at a position corresponding to amino acid position 197in SEQ ID NO: 1; vvv) an isoleucine (I) at a position corresponding toamino acid position 200 in SEQ ID NO: 1; www) a valine (V) at a positioncorresponding to amino acid position 203 in SEQ ID NO: 1; xxx) a leucine(L) at a position corresponding to amino acid position 204 in SEQ ID NO:1; yyy) a cysteine (C) at a position corresponding to amino acidposition 209 in SEQ ID NO: 1; zzz) a leucine (L) at a positioncorresponding to amino acid position 222 in SEQ ID NO: 1; aaaa) anisoleucine (I) at a position corresponding to amino acid position 232 inSEQ ID NO: 1; bbbb) a serine (S) at a position corresponding to aminoacid position 236 in SEQ ID NO: 1; cccc) a leucine (L) or an arginine(R) at a position corresponding to amino acid position 237 in SEQ ID NO:1; dddd) a histidine (H) at a position corresponding to amino acidposition 246 in SEQ ID NO: 1; eeee) an isoleucine (I) at a positioncorresponding to amino acid position 254 in SEQ ID NO: 1; ffff) a serine(S) at a position corresponding to amino acid position 258 in SEQ ID NO:1; gggg) an arginine (R) at a position corresponding to amino acidposition 267 in SEQ ID NO: 1; hhhh) an isoleucine (I) at a positioncorresponding to amino acid position 278 in SEQ ID NO: 1; iiii) atyrosine (Y) at a position corresponding to amino acid position 281 inSEQ ID NO: 1; jjjj) a phenylalanine (F) at a position corresponding toamino acid position 282 in SEQ ID NO: 1; kkkk) a threonine (T) at aposition corresponding to amino acid position 289 in SEQ ID NO: 1; llll)a glycine (G) at a position corresponding to amino acid position 308 inSEQ ID NO: 1; mmmm) an arginine (R) at a position corresponding to aminoacid position 311 in SEQ ID NO: 1; nnnn) an alanine (A) at a positioncorresponding to amino acid position 312 in SEQ ID NO: 1; oooo) analanine (A) at a position corresponding to amino acid position 316 inSEQ ID NO: 1; pppp) an arginine (R) at a position corresponding to aminoacid position 318 in SEQ ID NO: 1 qqqq) an alanine (A) at a positioncorresponding to amino acid position 334 in SEQ ID NO: 1; rrrr) aphenylalanine (F) at a position corresponding to amino acid position 339in SEQ ID NO: 1; ssss) a glycine (G) or a leucine (L) at a positioncorresponding to amino acid position 340 in SEQ ID NO: 1; tttt) a serine(S) at a position corresponding to amino acid position 342 in SEQ ID NO:1; uuuu) an asparagine (N) at a position corresponding to amino acidposition 345 in SEQ ID NO: 1; vvvv) an asparagine (N) at a positioncorresponding to amino acid position 346 in SEQ ID NO: 1; wwww) anasparagine (N) at a position corresponding to amino acid position 348 inSEQ ID NO: 1; or, xxxx) any combination of a) to wwww).

In another embodiment, the invention concerns the isolated orrecombinant polynucleotide of embodiment 1, and its correspondingpolypeptide, wherein said nucleotide sequence encodes a meganucleasepolypeptide, wherein said polypeptide further comprises at least oneamino acid modification described herein such as those shown in FIG.5A-FIG. 5E, FIG. 9A-FIG. 9N, FIG. 10A-FIG. 10D, FIG. 11, FIG. 12, FIG.13, FIG. 14A-FIG. 14F and FIG. 15A-FIG. 15E as well any I-Cre1 typemodification known and any combination thereof.

In another embodiment, the invention concerns an isolated or recombinantpolynucleotide, and its corresponding polypeptide, wherein saidnucleotide sequence encodes a meganuclease polypeptide selected from thegroup consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 251, 252, 253, 272, 273, 274, 275, 272,273, 274, 275, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,295, 296, 297, 298, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339,340, 341, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368,369, 370, 371, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400,401, 402, 403, 430, 431, 432 and 433.

In another embodiment, the invention concerns an isolated or recombinantpolynucleotide of the present disclosure, and its correspondingpolypeptide, wherein said nucleotide sequence encodes a meganucleasepolypeptide, wherein the polypeptide is capable of recognizing andcleaving a meganuclease recognition sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 85, SEQ ID NO: 269, SEQ ID NO:281, SEQ ID NO: 331, SEQ ID NO: 358, SEQ ID NO: 390, SEQ ID NO: 423 orSEQ ID NO: 424.

In another embodiment, the invention concerns an isolated or recombinantpolynucleotide of the present disclosure, and its correspondingpolypeptide, wherein said nucleotide sequence encodes a meganucleasepolypeptide, wherein said polypeptide has an increased meganucleaseactivity when compared to a control meganuclease that lacks said aminoacid modification. The control meganuclease can be selected from thegroup of SEQ ID NO: 1, SEQ ID NO: 86, SEQ ID NO: 250, SEQ ID NO: 270,SEQ ID NO: 271, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 329, SEQ IDNO: 356, SEQ ID NO: 389, SEQ ID NO: 429 or SEQ ID NO: 435 or any I-CreItype meganuclease. Increased meganuclease activity can be evidenced byany method for measuring meganuclease activity, including but notlimited to a) a higher yeast assay score when compared to the controlmeganuclease that lacks said amino acid modification; or, b) a highertarget site mutation rate when compared to the control meganuclease thatlacks said amino acid modification; or, c) a higher in-vitro cuttingwhen compared to the control meganuclease that lacks said amino acidmodification; or, d) any combination of those methods. Furthermore,increased activity can be measured at a wide range of temperatures suchas temperatures including 16° C., 24° C., 28° C., 30° C. or 37° C. andtemperatures between 16° C. to 37° C.

In another embodiment, the invention concerns an isolated or recombinantpolynucleotide, further comprising a nucleotide sequence encoding aN-terminal nuclear transit peptide and/or a nucleotide sequence encodinga C-terminal histidine tag.

In another embodiment, the invention concerns a recombinant DNAconstruct, comprising the isolated or recombinant polynucleotide of thepresent disclosure. The recombinant DNA construct can further comprise apromoter operably linked to said polynucleotide. The promoter can beheterologous with respect to the recombinant polynucleotide.

In another embodiment, the invention concerns a cell, plant cell, yeastcell, plant, yeast or seed comprising the recombinant construct of thepresent disclosure. The plant cell can be a monocot or a dicot plantcell. The monocot plant cell can be from maize, wheat, rice, barley,sugarcane, sorghum, or rye. The dicot cell can be a from soybean,Brassica, sunflower, cotton, or alfalfa.

In another embodiment, the invention concerns plants comprising therecombinant construct of the present disclosure and seeds or plantextracts, explant obtained from such plants.

In another embodiment, the invention concerns a method for producing ameganuclease having increased activity over a range of temperatures, themethod comprising:

-   -   a) producing a variant meganuclease by modifying at least one        amino acid at an amino acid position corresponding to a position        of SEQ ID NO: 1 selected from the group consisting of positions        2, 12, 16, 22, 23, 31, 36, 43, 50, 56, 58, 59, 62, 71, 72, 73,        80, 81, 82, 86, 91, 95, 98, 103, 113, 114, 116, 117, 118, 121,        124, 128, 129, 131, 147, 151, 153, 159, 160, 161, 162, 163, 164,        165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,        178, 179, 180, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,        192, 194, 195, 196, 197, 200, 203, 204, 209, 222, 232, 236, 237,        246, 254, 258, 267, 278, 281, 282, 289, 308, 311, 312, 316, 318,        319, 334, 339, 340, 342, 345, 346 348 and combinations thereof;        and,    -   b) selecting said variant meganuclease from step a) and        screening said variant meganuclease for the ability to cleave a        DNA target sequence over a range of temperatures between and        including 16° C. to 37° C.

In another embodiment, the invention concerns a method for producing ameganuclease having an increased meganuclease activity when compared toa control meganuclease, the method comprising:

-   -   a) producing a variant meganuclease by modifying at least one        amino acid at an amino acid position corresponding to a position        of SEQ ID NO: 1 selected from the group consisting of positions        2, 12, 16, 22, 23, 31, 36, 43, 50, 56, 58, 59, 62, 71, 72, 73,        80, 81, 82, 86, 91, 95, 98, 103, 113, 114, 116, 117, 118, 121,        124, 128, 129, 131, 147, 151, 153, 159, 160, 161, 162, 163, 164,        165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,        178, 179, 180, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,        192, 194, 195, 196, 197, 200, 203, 204, 209, 222, 232, 236, 237,        246, 254, 258, 267, 278, 281, 282, 289, 308, 311, 312, 316, 318,        319, 334, 339, 340, 342, 345, 346, 348 and combinations thereof;        and,    -   b) selecting the variant meganuclease from step a) and screening        said variant for increased meganuclease activity when compared        to a control meganuclease.        In another embodiment, the invention concerns a method of        introducing a double-strand break in the genome of a yeast or        plant cell, said method comprising:

a) contacting at least one plant or yeast cell comprising in its genomea meganuclease recognition site with a variant meganuclease polypeptideselected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 251, 252, 253, 272,273, 274, 275, 272, 273, 274, 275, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 330, 331, 332, 333, 334, 335,336, 337, 338, 339, 340, 341, 357, 358, 359, 360, 361, 362, 363, 364,365, 366, 367, 368, 369, 370, 371, 390, 391, 392, 393, 394, 395, 396,397, 398, 399, 400, 401, 402 and 403, wherein the variant meganucleaseis capable of inducing a double-strand break in said recognition site;and,

-   -   b) selecting the yeast or plant cell from a) and screening said        yeast or plant cell for any modification of said recognition        sequence.

In another embodiment, the invention concerns a method of integrating apolynucleotide of interest into a recognition site in the genome of aplant or yeast cell, the method comprising:

a) contacting at least one plant or yeast cell comprising in its genomea meganuclease recognition site with:

-   -   (i) a variant meganuclease polypeptide selected from the group        consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22,        23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,        87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,        102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,        115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,        128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,        141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,        154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,        167, 251, 252, 253, 272, 273, 274, 275, 272, 273, 274, 275, 284,        285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,        298, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341,        357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369,        370, 371, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400,        401, 402 and 403,    -   wherein the variant meganuclease is capable of inducing a        double-strand break in said recognition site; and,    -   (ii) a DNA fragment containing a polynucleotide of interest;

b) selecting at least one plant or yeast cell comprising integration ofthe polynucleotide of interest cassette at the recognition site.

In another embodiment, the invention concerns an isolated or recombinantpolynucleotide, and its corresponding polypeptide, encoding ameganuclease polypeptide, said polypeptide comprising an amino acidsequence having at least one amino acid modification at an amino acidposition corresponding to a position of SEQ ID NO: 1 selected from thegroup consisting of positions 16, 22, 50, 56, 59, 71, 81, 103, 121, 153,185, 209, 222, 246, 258, 281, 308, 316, 345, 346, and combinationsthereof, and wherein the polypeptide is capable of recognizing andcleaving a meganuclease target site comprising SEQ ID NO: 2.

In another embodiment, the invention concerns an isolated or recombinantpolynucleotide encoding a meganuclease polypeptide, the polypeptidecomprising an amino acid sequence having at least one amino acidmodification at an amino acid position corresponding to a position ofSEQ ID NO: 86 selected from the group consisting of positions 2, 12, 16,22, 23, 36, 43, 50, 56, 58, 59, 72, 73, 81, 86, 91, 95, 103, 113, 114,120, 121, 124, 128, 129, 131, 151, 153, 200, 204, 209, 232, 236, 237,246, 254, 258, 267, 281, 308, 311, 312, 316, 319, 334, 339, 340, 342,and combinations thereof, and wherein the polypeptide is capable ofrecognizing and cleaving a meganuclease target site comprising SEQ IDNO: 85.

In another embodiment, the invention concerns an isolated or recombinantpolynucleotide encoding a meganuclease polypeptide, the polypeptidecomprising an amino acid sequence having at least one amino acidmodification at an amino acid position corresponding to a position ofSEQ ID NO: 270 selected from the group consisting of positions 16, 22,50, 71, 185, 246, 258, 316 and combinations thereof, and wherein thepolypeptide is capable of recognizing and cleaving a meganuclease targetsite comprising SEQ ID NO: 269.

In another embodiment, the invention concerns an isolated or recombinantpolynucleotide encoding a meganuclease polypeptide, the polypeptidecomprising an amino acid sequence having at least one amino acidmodification at an amino acid position corresponding to a position ofSEQ ID NO: 329 selected from the group consisting of positions 12, 32,50, 56, 80, 105, 124, 129, 131, 153, 185, 311, 316, 318, 340, andcombinations thereof, and wherein the polypeptide is capable ofrecognizing and cleaving a meganuclease target site comprising SEQ IDNO: 328.

In another embodiment, the invention concerns an isolated or recombinantpolynucleotide encoding a meganuclease polypeptide, the polypeptidecomprising an amino acid sequence having at least one amino acidmodification at an amino acid position corresponding to a position ofSEQ ID NO: 356 selected from the group consisting of positions 12, 24,36, 50, 56, 62, 73, 80, 124, 129, 147, 182, 203, 237, 252, 311, 316,318, 340, 348, and combinations thereof, and wherein the polypeptide iscapable of recognizing and cleaving a meganuclease target sitecomprising SEQ ID NO: 355.

In another embodiment, the invention concerns an isolated or recombinantpolynucleotide encoding a meganuclease polypeptide, the polypeptidecomprising an amino acid sequence having at least one amino acidmodification at an amino acid position corresponding to a position ofSEQ ID NO: 389 selected from the group consisting of positions 12, 50,56, 124, 129, 131, 153, 211, 237, 311, 316, and position 318, andcombinations thereof, and wherein the polypeptide is capable ofrecognizing and cleaving a meganuclease target site comprising SEQ IDNO: 388.

In another embodiment, the invention concerns An isolated or recombinantpolynucleotide encoding a meganuclease polypeptide, the polypeptidecomprising an amino acid sequence having at least one amino acidmodification at an amino acid position corresponding to a position ofSEQ ID NO: 429 selected from the group consisting of positions 16, 22,50, 71, 185, 246, 258, 316 and combinations thereof, and wherein thepolypeptide is capable of recognizing and cleaving a meganuclease targetsite comprising SEQ ID NO: 423.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing, whichform a part of this application. The sequence descriptions and sequencelisting attached hereto comply with the rules governing nucleotide andamino acid sequence disclosures in patent applications as set forth in37 C.F.R. §§1.821 1.825. The sequence descriptions contain the threeletter codes for amino acids as defined in 37 C.F.R. §§1.821 1.825,which are incorporated herein by reference.

FIG. 1A-FIG. 1B show an amino acid alignment of I-CreI meganuclease(I-CreI.pro, SEQ ID NO: 3) with related meganucleases (SEQ ID NOs: 4-13)from various species. The decoration shows amino acid residues sharingidentity.

FIG. 2 shows a diagram representing the yeast screening system used todetermine the meganuclease activity in yeast. Gene fragmentscorresponding to the first 1000 nucleotides of the yeast Ade2 codingsequence (Ade2 5′ fragment) and the last 1011 nucleotides of the yeastAde2 coding sequence (Ade2 3′ fragment) were disrupted by a fragmentincluding the yeast ura3 gene (Ura3) and meganuclease recognition sitesfor I-SceI.

FIG. 3 shows the numerical scale and corresponding white sectoring ofyeast colonies used to quantify meganuclease activity. Since thesectoring phenotype is a qualitative measure of meganuclease activity, a0-4 numerical scoring system was implemented. A score of 0 indicatesthat no white sectors (no meganuclease cutting) were observed; a scoreof 4 indicates completely white colonies (complete cutting of therecognition site); scores of 1-3 indicate intermediate white sectoringphenotypes (and intermediate degrees of recognition site cutting).

FIG. 4 shows the meganuclease expression plasmid pVER8134.

FIG. 5A-FIG. 5E show an amino acid alignment of the parental LIG3-4(LIG3-4.pro, SEQ ID NO: 1) and LIG3-4 meganuclease variants (Table 1A,SEQ ID NOs: 14-38). The name of the meganuclease listed in FIG. 5A-FIG.5E corresponds to the name in Table 1A but include a “.pro” to indicatethat this is a protein alignment.

FIG. 6A-FIG. 6C show the percent cleavage by the parental LIG3-4 andLIG3-4 variant meganucleases (B65=LIG3-4(B65); hit15=LIG3-4(15);hit7=LIG3-4(7)) of plasmid DNA substrate at 0, 25, 50 and 75 minutesaveraged across three replicates of real-time PCR. FIG. 6A shows the %cleavage observed at 23° C. FIG. 6B shows the % cleavage observed at 28°C. FIG. 6C shows the % cleavage observed at 37° C.

FIG. 7A-FIG. 7C show the percent cleavage by the parental LIG3-4 andLIG3-4 variant meganucleases (B65=LIG3-4(B65); hit15=LIG3-4(15);hit7=LIG3-4(7)) of genomic DNA substrate at 50 minutes averaged acrossthree replicates of real-time PCR. FIG. 7A shows the % cleavage observedat 23° C. FIG. 7B shows the % cleavage observed at 28° C. FIG. 7C showsthe % cleavage observed at 37° C.

FIG. 8A shows a schematic outline of long fragment PCR reactions used toconfirm UBI:moPAT:PinII cassette integration at the endogenous LIG3-4recognition site. FIG. 8B: shows the results of long fragment PCR oncallus from four events where integration occurred at the recognitionsite. The left panel of FIG. 8B shows the long junction fragment PCR onthe HR1 side using genomic primer (HRR1) and moPAT primer (mopatR2); Theright panel of FIG. 8B shows the long junction fragment PCR on HR2 side(mopatF2/HR2R2). Primer set mopatF2/HR2R2 amplified a 4 kb fragment,spanning from moPAT gene through the UBI intron, UBI promoter, and theHR2 sequence to the adjacent genomic region. Primer set HRR1/mopatR2amplified a 2.2 kb fragment, spanning from the moPAT gene through theHR1 to the adjacent genomic region. The sizes of the two long PCRproducts indicate a perfect integration of the donor gene cassette atLIG3-4 recognition site. Insertion was obtained in T0 and T1 plants fromone of the callus event.

FIG. 9A-FIG. 9N show an amino acid alignment of the parental MHP77 (SEQID NO: 86) and MHP77 meganucleases variants. (Table 1A, SEQ ID NOs:87-167). The name of the meganuclease listed in FIG. 9A-FIG. 9Ncorresponds to the name in Table 1A. Amino acid modifications of thevariant meganucleases, when compared to the parental meganuclease MHP77,are shown. A (-) indicates that the amino acid residue of the variantand parental meganuclease were identical.

FIG. 10A-FIG. 10D show an amino acid alignment of the parental MHP14(SEQ ID NO:282) and MHP14 meganuclease variants. (Table 1B, SEQ IDNOs:284-298). The name of the meganuclease listed in FIG. 10A-FIG. 10Dcorresponds to the name in Table 1B. Amino acid modifications of thevariant meganucleases, when compared to the parental meganuclease MHP14,are shown. A (-) indicates that the amino acid residue of the variantand parental meganuclease were identical.

FIG. 11 provides an amino acid alignment of the parental MHP107 (SEQ IDNO:329) and MHP107 meganucleases variants Table 1C, SEQ ID NOs:330-341).The name of the meganuclease listed in FIG. 11 corresponds to the namein Table 1C. Amino acid modifications of the variant meganucleases, whencompared to the parental meganuclease, are shown. A (-) indicates thatthe amino acid residue of the variant and parental meganuclease wereidentical.

FIG. 12 provides an amino acid alignment of the parental ZM6.3 (SEQ IDNo:356) and ZM6.3 meganucleases variants Table 1D, SEQ ID NOs:357-371).The name of the meganuclease listed in FIG. 12 corresponds to the namein Table 1D. Amino acid modifications of the variant meganucleases, whencompared to the parental meganuclease, are shown. A (-) indicates thatthe amino acid residue of the variant and parental meganuclease wereidentical.

FIG. 13 provides an amino acid alignment of the parental ZM6.22v2 (SEQID NO:389) and ZM6.22v2 meganucleases variants Table 1E. SEQ IDNOs:390-403). The name of the meganuclease listed in FIG. 12 correspondsto the name in Table 1E. Amino acid modifications of the variantmeganucleases, when compared to the parental meganuclease, are shown. A(-) indicates that the amino acid residue of the variant and parentalmeganuclease were identical.

FIG. 14A-FIG. 14F show an amino acid alignment of the LIG3-4meganuclease (SEQ ID NO: 1) and multiple meganucleases (The name of themeganuclease listed in FIG. 14A-FIG. 14F corresponds to the name inTable 1A-1E and corresponding SEQ ID NOs are shown in Table 1A-1E).Amino acid modifications different from SEQ ID NO: 1 are shown. A (-)indicates that the amino acid residue of the meganuclease is identicalto the LIG3-4 meganuclease (SEQ ID NO: 1). Highlighted in gray aremutations which were correlated with increased meganuclease activity onthe desired target site.

FIG. 15A-FIG. 15D show an amino acid alignment of some meganucleasescomprising a linker polypeptide that links the two re-engineered I-CreImonomers into a single amino chain. FIG. 15 E shows the percent identifyof some variant (MHP14(10), MHP77(L9-01) and parental (LIG3-4, MHP14,MHP77) meganucleases). Highlighted in gray are the novel linkersequences present in variants MHP14(10) and MHP77(L9-01).

FIG. 16 shows the structural motives of the meganuclease. Alpha helix-1encompasses amino acids 8 through 19 on subunit number 1and amino acids195 through 206 on subunit number 2 in SEQ ID NO: 1. Alpha helix-5encompasses amino acids 120-135 on subunit number 1 and amino acids 307through 322 on subunit number 2 in SEQ ID NO: 1 .

SEQUENCES

SEQ ID NO: 1 is the amino acid sequence of the single chain LIG3-4meganuclease fusion polypeptide.

SEQ ID NO: 2 is the nucleotide sequence of the LIG3-4 recognitionsequence.

SEQ ID NO: 3 is the amino acid sequence of the I-CreI meganucleasemonomer.

SEQ ID NO: 4 is the amino acid sequence of gi_18654305

SEQ ID NO: 5 is the amino acid sequence of gi_108773071

SEQ ID NO: 6 is the amino acid sequence of gi_108773352

SEQ ID NO: 7 is the amino acid sequence of gi_108796958

SEQ ID NO: 8 is the amino acid sequence of gi_12667512

SEQ ID NO: 9 is the amino acid sequence of gi_18654311

SEQ ID NO: 10 is the amino acid sequence of gi_150406493

SEQ ID NO: 11 is the amino acid sequence of gi_110225678

SEQ ID NO: 12 is the amino acid sequence of gi_11467050

SEQ ID NO: 13 is the amino acid sequence of gi_18654162

SEQ ID NO: 14 is the amino acid sequence of the LIG3-4 meganuclease.

TABLE 1A Listing of SEQ ID NO: s (NT = nucleotide sequence; AA = aminoacid sequence) for parental and variant meganucleases. DNA forexpression AA in yeast Name SEQ ID NO: SEQ ID NO: LIG3-4 1 40LIG3-4(B65) 27 54 LIG3-4(B70) 28 55 LIG3-4(B75) 31 58 LIG3-4(B76) 32 59LIG3-4(B73) 30 57 LIG3-4(B82) 34 61 LIG3-4(B78) 33 60 LIG3-4(B1) 18 45LIG3-4(15) 15 42 LIG3-4(D8) 38 65 LIG3-4(B15) 19 46 LIG3-4(C1) 35 62LIG3-4(B71) 29 56 LIG3-4(B39) 24 51 LIG3-4(B16) 20 47 LIG3-4(D7) 37 64LIG3-4(B38) 23 50 LIG3-4(B40) 25 52 LIG3-4(B36) 22 49 LIG3-4(B24) 21 48LIG3-4(B55) 26 53 LIG3-4(A4) 16 43 LIG3-4(D5) 36 63 LIG3-4(7) 14 41LIG3-4(A6) 17 44 MHP77 86 168 MHP77(L72-01a) 87 169 MHP77(L72-08a) 88170 MHP77(L72-09a) 89 171 MHP77(L73-02a) 90 172 MHP77(L73-05a) 91 173MHP77(L9-01) 92 174 MHP77(L9-02) 93 175 MHP77(L9-03) 94 176 MHP77(L9-04)95 177 MHP77(L9-06) 96 178 MHP77(L9-09) 97 179 MHP77(L9-10) 98 180MHP77(L9-11) 99 181 MHP77(L9-12) 100 182 MHP77(L112-03a) 101 183MHP77(L113-01) 102 184 MHP77(L13-01a) 103 185 MHP77(L13-02) 104 186MHP77(L13-04) 105 187 MHP77(L13-06) 106 188 MHP77(L13-08a) 107 189MHP77(L13-10B1) 108 190 MHP77(L13-11) 109 191 MHP77(L13-12) 110 192MHP77(L15-02) 111 193 MHP77(L15-03) 112 194 MHP77(L15-05) 113 195MHP77(L15-06) 114 196 MHP77(L15-08) 115 197 MHP77(L15-10) 116 198MHP77(L15-11) 117 199 MHP77(L15-12) 118 200 MHP77(L15-13) 119 201MHP77(L15-15) 120 202 MHP77(L15-16) 121 203 MHP77(L15-18) 122 204MHP77(L15-20) 123 205 MHP77(L15-21) 124 206 MHP77(L15-23) 125 207MHP77(L15-24) 126 208 MHP77(L15-28) 127 209 MHP77(L15-29) 128 210MHP77(L15-33) 129 211 MHP77(L15-34) 130 212 MHP77(L15-35) 131 213MHP77(L15-36) 132 214 MHP77(L15-39) 133 215 MHP77(L15-40) 134 216MHP77(L15-41) 135 217 MHP77(L15-42) 136 218 MHP77(L15-43) 137 219MHP77(L15-45) 138 220 MHP77(L15-46) 139 221 MHP77(L15-27) 140 222MHP77(L15-30) 141 223 MHP77(L15-31) 142 224 MHP77(L15-47) 143 225MHP77(L16-01) 144 226 MHP77(L16-02) 145 227 MHP77(L16-03) 146 228MHP77(L16-04) 147 229 MHP77(L16-05) 148 230 MHP77(L16-06) 149 231MHP77(L16-07) 150 232 MHP77(L16-08) 151 233 MHP77(L16-09) 152 234MHP77(L16-11) 153 235 MHP77(L16-12) 154 236 MHP77(L16-14) 155 237MHP77(L16-15) 156 238 MHP77(L16-16) 157 239 MHP77(L16-17) 158 240MHP77(L16-18) 159 241 MHP77(L16-19) 160 242 MHP77(L16-21) 161 243MHP77(L16-23) 162 244 MHP77(L16-24) 163 245 MHP77(L17-12) 164 246MHP77(L18-01) 165 247 MHP77(L18-12) 166 248 MHP77(L17-01) 167 249

SEQ ID NO: 39 is the plant optimized nucleotide sequence of LIG3-4comprising a nuclear localization signal and an intron.

SEQ ID NO: 66 is the nucleotide sequence of MN031 primer.

SEQ ID NO: 67 is the nucleotide sequence of MN022 primer.

SEQ ID NO: 68 is the nucleotide sequence of plasmid pVER8134.

SEQ ID NO: 69 is the nucleotide sequence of a nuclear localizationsignal.

SEQ ID NO: 70 is the amino acid sequence of a nuclear localizationsignal.

SEQ ID NO: 71 is the amino acid sequence of 6× histidine tag.

SEQ ID NO: 72 is the nucleotide sequence of a nuclear localizationsignal in maize.

SEQ ID NO: 73 is the plant-optimized nucleotide sequence of theLIG3-4(7) meganuclease with a nuclear localization signal and an intron.

SEQ ID NO: 74 is the plant-optimized nucleotide sequence of theLIG3-4(15) meganuclease with a nuclear localization signal and anintron.

SEQ ID NO: 75 is the plant-optimized nucleotide sequence of theLIG3-4(B65) meganuclease with a nuclear localization signal and anintron.

SEQ ID NO: 76 is the nucleotide sequence of plasmid PHP46961.

SEQ ID NO: 77 is the nucleotide sequence of LIG3-4(HR1).

SEQ ID NO: 78 is the nucleotide sequence of LIG3-4(HR2).

SEQ ID NO: 79 is the nucleotide sequence of LIG3-4 target site qPCRprobe.

SEQ ID NO: 80 is the nucleotide sequence of Lig3-4_forward primer.

SEQ ID NO: 81 is the nucleotide sequence of Lig3-4_reverse primer.

SEQ ID NO: 82 is the nucleotide sequence of yeast ade2.

SEQ ID NO: 83 is the nucleotide coding sequence of ade2.

SEQ ID NO: 84 is the nucleotide sequence of plasmid pHD1327.

SEQ ID NO: 85 is the nucleotide sequence the MHP77 recognition site.

SEQ ID NO: 86 is the amino acid sequence of the MHP77 meganuclease.

SEQ ID NO: 250 is the amino acid sequence of the MHP77.3 meganuclease.

SEQ ID NO: 251 is the amino acid sequence of the MHP77.3 (L9-02)meganuclease.

SEQ ID NO: 252 is the amino acid sequence of the MHP77.3 (L9-11)meganuclease.

SEQ ID NO: 253 is the amino acid sequence of the MHP77.3 (L9-12)meganuclease.

SEQ ID NO: 254 is the plant-optimized nucleotide sequence of MHP77comprising a nuclear localization signal and lacking an intron.

SEQ ID NO: 255 is the plant-optimized nucleotide sequence of MHP77.3meganuclease MHP77 comprising a nuclear localization signal and lackingan intron.

SEQ ID NO: 256 is the plant-optimized nucleotide sequence ofMHP77(L9-02) meganuclease comprising a nuclear localization signal andan intron.

SEQ ID NO: 257 is the plant-optimized nucleotide sequence of the MHP77(L9-11) meganuclease comprising a nuclear localization signal and anintron.

SEQ ID NO: 258 is the plant-optimized nucleotide sequence of the MHP77(L9-12) meganuclease comprising a nuclear localization signal and anintron

SEQ ID NO: 259 is the plant-optimized nucleotide sequence of MHP77.3(L9-02) meganuclease comprising a nuclear localization signal and anintron.

SEQ ID NO: 260 is the plant-optimized nucleotide sequence of the MHP77.3(L9-11) meganuclease comprising a nuclear localization signal and anintron.

SEQ ID NO: 261 is the plant-optimized nucleotide sequence of the MHP77.3(L9-12) meganuclease comprising a nuclear localization signal and anintron SEQ ID NO: 262 is the amino acid sequence of the MHP77.3(15)meganuclease.

SEQ ID NO: 263 is the plant-optimized nucleotide sequence of MHP77.3(15)meganuclease comprising a nuclear localization signal and an intron.

SEQ ID NO: 264 is the nucleotide sequence of the MHP77HR1.

SEQ ID NO: 265 is the nucleotide sequence of the MHP77HR2.

SEQ ID NO: 266 is the nucleotide sequence of the MHP77 target site qPCRprobe.

SEQ ID NO: 267 is the nucleotide sequence of the MHP77_forward primer.

SEQ ID NO: 268 is the nucleotide sequence of the MHP77_reverse primer.

SEQ ID NO: 269 is the nucleotide sequence of the MS26 recognition site.

SEQ ID NO: 270 is the amino acid sequence of the MS26+ meganuclease.

SEQ ID NO: 271 is the amino acid sequence of the MS26++ meganuclease.

SEQ ID NO: 272 is the amino acid sequence of the MS26+(7) meganuclease.

SEQ ID NO: 273 is the amino acid sequence of the MS26+(15) meganuclease.

SEQ ID NO: 274 is the amino acid sequence of the MS26+(B65)meganuclease.

SEQ ID NO: 275 is the amino acid sequence of the MS26++(15)meganuclease.

SEQ ID NO: 276 is the plant-optimized nucleotide sequence of MS26+ andno intron

SEQ ID NO: 419 is the plant-optimized nucleotide sequence of MS26+(7)and no intron

SEQ ID NO: 277 is the plant-optimized nucleotide sequence of MS26+(15)and no intron

SEQ ID NO: 278 is the plant-optimized nucleotide sequence of MS26+(B65)and no intron;

SEQ ID NO: 279 is the plant-optimized nucleotide sequence of MS26++ andno intron

SEQ ID NO: 280 is the plant-optimized nucleotide sequence of MS26++(15)and no intron

SEQ ID NO: 281 is the nucleotide sequence of the MHP14 recognition site.

TABLE 1B Listing of SEQ ID NO: s (NT = nucleotide sequence; AA = aminoacid sequence) for parental and variant meganucleases. DNA forexpression AA in yeast Name SEQ ID NO: SEQ ID NO: MHP14 282 299 MHP14+283 MHP14(01) 284 300 MHP14(02) 285 301 MHP14(03) 286 302 MHP14(04) 287303 MHP14(06) 288 304 MHP14(07) 289 305 MHP14(08) 290 306 MHP14(09) 291307 MHP14(10) 292 308 MHP14(12) 293 309 MHP14(13) 294 310 MHP14(14) 295311 MHP14(L14-03) 296 312 MHP14(L14-04) 297 313 MHP14(L14-07) 298 314

SEQ ID NO: 315 is the amino acid sequence of the MHP14+(04)meganuclease.

SEQ ID NO: 316 is the amino acid sequence of the MHP14+(06)meganuclease.

SEQ ID NO: 317 is the amino acid sequence of the MHP14+(08)meganuclease.

SEQ ID NO: 318 is the amino acid sequence of the MHP14+(12)meganuclease.

SEQ ID NO: 319 is the amino acid sequence of the MHP14+(14)meganuclease.

SEQ ID NO: 320 is the amino acid sequence of the MHP14+(15)meganuclease.

SEQ ID NO: 321 is the plant-optimized nucleotide sequence of MHP14 andan intron.

SEQ ID NO: 322 is the plant-optimized nucleotide sequence of MHP14+(04)and an intron.

SEQ ID NO: 323 is the plant-optimized nucleotide sequence of MHP14+(06)and an intron.

SEQ ID NO: 324 is the plant-optimized nucleotide sequence of MHP14+(08)and an intron.

SEQ ID NO: 325 is the plant-optimized nucleotide sequence of MHP14+(12)and an intron.

SEQ ID NO: 326 is the plant-optimized nucleotide sequence of MHP14+(14)and an intron.

SEQ ID NO: 327 is the plant-optimized nucleotide sequence of MHP14+(15)and an intron.

SEQ ID NO: 328 is the nucleotide sequence of the MHP107 recognitionsite.

TABLE 1C Listing of SEQ ID NO: s (NT = nucleotide sequence; AA = aminoacid sequence) for parental and variant meganucleases. DNA forexpression AA in yeast Name SEQ ID NO: SEQ ID NO: MHP107 329 342MHP107(C1) 330 343 MHP107(C2) 331 344 MHP107(C3) 332 345 MHP107(C4) 333346 MHP107(C5) 334 347 MHP107(C6) 335 348 MHP107(D2) 336 349 MHP107(D3)337 350 MHP107(D4) 338 351 MHP107(D5) 339 352 MHP107(D1) 340 353MHP107(D6) 341 354

SEQ ID NO: 355 is the nucleotide sequence of the ZM6.3 recognition site.

TABLE 1D Listing of SEQ ID NO: s (NT = nucleotide sequence; AA = aminoacid sequence) for parental and variant meganucleases. DNA forexpression AA in yeast Name SEQ ID NO: SEQ ID NO: ZM6.3 356 372ZM6.3(G1) 357 373 ZM6.3(G2) 358 374 ZM6.3(G3) 359 375 ZM6.3(G4) 360 376ZM6.3(G5) 361 377 ZM6.3(G6) 362 378 ZM6.3(H1) 363 379 ZM6.3(H2) 364 380ZM6.3(H3) 365 381 ZM6.3(H5) 366 382 ZM6.3(H6) 367 383 ZM6.3(1) 368 384ZM6.3(3) 369 385 ZM6.3(4) 370 386 ZM6.3(5) 371 387

SEQ ID NO: 388 is the nucleotide sequence of the ZM6.22v2 recognitionsite.

TABLE 1E Listing of SEQ ID NO: s (NT = nucleotide sequence; AA = aminoacid sequence) for parental and variant meganucleases. DNA forexpression AA in yeast Name SEQ ID NO: SEQ ID NO: ZM6.22v2 389 404ZM6.22v2(J2) 390 405 ZM6.22v2(J3) 391 406 ZM6.22v2(J4) 392 407ZM6.22v2(J5) 393 408 ZM6.22v2(I2) 394 409 ZM6.22v2(I3) 395 410ZM6.22v2(I4) 396 411 ZM6.22v2(I5) 397 412 ZM6.22v2(I6) 398 413ZM6.22v2(I7) 399 414 ZM6.22v2(I8) 400 415 ZM6.22v2(I9) 401 416ZM6.22v2(J7) 402 417 ZM6.22v2(J8) 403 418

SEQ ID NO: 419 is the nucleotide sequence of the MS26+(7) variantmeganuclease with no intron

SEQ ID NO: 420 is the nucleotide sequence of the linker polypeptide ofLIG3-4, MHP14, MHP77.

SEQ ID NO: 421 is the nucleotide sequence of the linker polypeptide ofMHP14(10).

SEQ ID NO: 422 is the nucleotide sequence of the linker polypeptide ofMHP77(L9-01) SEQ ID NO: 423 is the nucleotide sequence of the TS21recognition site in soybean genome.

SEQ ID NO: 424 is the nucleotide sequence of the TS14 recognition sitein soybean genome.

SEQ ID NO: 425 is the plant-optimized nucleotide sequence of the TS21meganuclease with a nuclear localization signal and an intron.

SEQ ID NO: 426 is the plant-optimized nucleotide sequence of the TS21(7)meganuclease with a nuclear localization signal and an intron.

SEQ ID NO: 427 is the plant-optimized nucleotide sequence of theTS21(15) meganuclease with a nuclear localization signal and an intron.

SEQ ID NO: 428 plant-optimized nucleotide sequence of the TS21(B65)meganuclease with a nuclear localization signal and an intron.

SEQ ID NO: 429 is the amino acid sequence of the TS21 meganuclease.

SEQ ID NO: 430 is the amino acid sequence of the TS21(7) meganuclease.

SEQ ID NO: 431 is the amino acid sequence of the TS21(15) meganuclease.

SEQ ID NO: 432 is the amino acid sequence of the TS21(B65) meganuclease

SEQ ID NO: 433 is the plant-optimized nucleotide sequence of TS14meganuclease with a nuclear localization signal and an intron.

SEQ ID NO: 434 is the plant-optimized nucleotide sequence of TS14(15)meganuclease with a nuclear localization signal and an intron

SEQ ID NO: 435 is the amino acid sequence of the TS14 meganuclease.

SEQ ID NO: 436 is the amino acid sequence of the TS14(15) meganuclease.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants; reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, specific examples ofappropriate materials and methods are described herein.

In the context of this disclosure, a number of terms and abbreviationsare used. The following definitions are provided.

I. Overview

Compositions and methods comprising polynucleotides and polypeptideshaving meganuclease activity are provided. Also provided arecompositions with increased meganuclease activity and methods of use.Further provided are nucleic acid constructs, yeasts, plants, plantcells, explants, seeds and grain having the meganuclease sequences. Themethods and compositions employ endonucleases capable of inducing adouble-strand break at a recognition sequence within a DNA fragment orwithin the genome of a yeast cell, plant, plant cell or seed.

II. Compositions

As used herein, an “isolated” polynucleotide or polypeptide, orbiologically active portion thereof, is substantially or essentiallyfree from components that normally accompany or interact with thepolynucleotide or polypeptide as found in its naturally occurringenvironment. Thus, an isolated or purified polynucleotide or polypeptideis substantially free of other cellular material or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. Optimally, an“isolated” polynucleotide is free of sequences (optimally proteinencoding sequences) that naturally flank the polynucleotide (i.e.,sequences located at the 5′ and 3′ ends of the polynucleotide) in thegenomic DNA of the organism from which the polynucleotide is derived.For example, in various embodiments, the isolated polynucleotide cancontain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kbof nucleotide sequence that naturally flank the polynucleotide ingenomic DNA of the cell from which the polynucleotide is derived. Apolypeptide that is substantially free of cellular material includespreparations of polypeptides having less than about 30%, 20%, 10%, 5%,or 1% (by dry weight) of contaminating protein. When the polypeptide ofthe invention or biologically active portion thereof is recombinantlyproduced, optimally culture medium represents less than about 30%, 20%,10%, 5%, or 1% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals.

As used herein, polynucleotide or polypeptide is “recombinant” when itis artificial or engineered, or derived from an artificial or engineeredprotein or nucleic acid. For example, a polynucleotide that is insertedinto a vector or any other heterologous location, e.g., in a genome of arecombinant organism, such that it is not associated with nucleotidesequences that normally flank the polynucleotide as it is found innature is a recombinant polynucleotide. A polypeptide expressed in vitroor in vivo from a recombinant polynucleotide is an example of arecombinant polypeptide. Likewise, a polynucleotide sequence that doesnot appear in nature, for example, a variant of a naturally occurringgene is recombinant.

A “subsequence” or “fragment” is any portion of an entire sequence.

The terms “target site”, “target sequence”, “genomic target site” and“genomic target sequence” are used interchangeably herein and refer to apolynucleotide sequence in the genome of a plant cell or yeast cell thatcomprises a recognition sequence for a double-strand break inducingagent.

As used herein, the term “recognition sequence” refers to a DNA sequenceat which a double-strand break is induced in the plant cell genome by anendonuclease. The terms “recognition sequence”, “recognition site”,“recognition site for an endonuclease”, “meganuclease recognitionsequence” and “meganuclease recognition site” are used interchangeablyherein. The recognition site can be an endogenous site in the plantgenome, or alternatively, the recognition site can be heterologous tothe plant and thereby not be naturally occurring in the genome, or therecognition site can be found in a heterologous genomic locationcompared to where it occurs in nature. As used herein, the term“endogenous recognition site” refers to an endonuclease recognition sitethat is endogenous or native to the genome of a plant and is located atthe endogenous or native position of that recognition site in the genomeof the plant. The length of the recognition site can vary, and includes,for example, recognition sites that are at least 4, 6, 8, 10, 12, 14,16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70or more nucleotides in length. It is further possible that therecognition site could be palindromic, that is, the sequence on onestrand reads the same in the opposite direction on the complementarystrand. The nick/cleavage site could be within the recognition sequenceor the nick/cleavage site could be outside of the recognition sequence.In another variation, the cleavage could occur at nucleotide positionsimmediately opposite each other to produce a blunt end cut or, in othercases, the incisions could be staggered to produce single-strandedoverhangs, also called “sticky ends”, which can be either 5′ overhangs,or 3′ overhangs.

In one embodiment, the recognition sequence of the endonucleasecomprises the LIG3-4 (SEQ ID NO: 2), MHP77 (SEQ ID NO: 85), MS26 (SEQ IDNO: 269), MHP14 (SEQ ID NO: 281), MP107 (SEQ ID NO: 328), ZM6.3 (SEQ IDNO: 355) and/or ZM6.22V2 (SEQ ID NO: 388) recognition sites of maizeand/or the TS21 (SEQ ID NO: 423) and/or the TS14 (SEQ ID NO: 424)recognition sites of soybean.

Active variants and fragments of the recognition can comprise at least65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity tothe given recognition sequence, wherein the active variants retainbiological activity and hence are capable of being recognized andcleaved by an endonuclease.

An “artificial target sequence” is a target sequence that has beenintroduced into the genome of a plant. Such an artificial targetsequence can be identical in sequence to an endogenous or native targetsequence in the genome of a plant but be located in a different position(i.e., a non-endogenous or non-native position) in the genome of aplant.

The terms “endogenous target sequence” and “native target sequence” areused interchangeable herein to refer to a target sequence that isendogenous or native to the genome of a plant and is at the endogenousor native position of that target sequence in the genome of the plant.

An “altered target sequence” refers to a target sequence that comprisesat least one alteration when compared to non-altered target sequence.Such “alterations” include, for example: (i) replacement of at least onenucleotide, (ii) a deletion of at least one nucleotide, (iii) aninsertion of at least one nucleotide, or (iv) any combination of(i)-(iii).

The term “double-strand-break-inducing agent” as used herein refers toany nuclease which produces a double-strand break in the targetsequence. Producing the double-strand break in a target sequence orother DNA can be referred to herein as “cutting” or “cleaving” thetarget sequence or other DNA.

An “endonuclease” refers to an enzyme that cleaves the phosphodiesterbond within a polynucleotide chain.

Endonucleases include restriction endonucleases that cleave DNA atspecific sites without damaging the bases. Restriction endonucleasesinclude Type I, Type II, Type III, and Type IV endonucleases, whichfurther include subtypes. In the Type I and Type III systems, both themethylase and restriction activities are contained in a single complex.Type I and Type III restriction endonucleases recognize specificrecognition sites, but typically cleave at a variable position from therecognition site, which can be hundreds of base pairs away from therecognition site. In Type II systems the restriction activity isindependent of any methylase activity, and cleavage typically occurs atspecific sites within or near to the recognition site. Most Type IIenzymes cut palindromic sequences, however Type IIa enzymes recognizenon-palindromic recognition sites and cleave outside of the recognitionsite, Type IIb enzymes cut sequences twice with both sites outside ofthe recognition site, and Type IIs enzymes recognize an asymmetricrecognition site and cleave on one side and at a defined distance ofabout 1-20 nucleotides from the recognition site. Type IV restrictionenzymes target methylated DNA. Restriction enzymes are further describedand classified, for example in the REBASE database (webpage atrebase.neb.com; Roberts et al., (2003) Nucleic Acids Res 31:418-20),Roberts et al., (2003) Nucleic Acids Res 31:1805-12, and Belfort et al.,(2002) in Mobile DNA II, pp. 761-783, Eds. Craigie et al., (ASM Press,Washington, D.C.).

An “engineered endonuclease” refers to an endonuclease that isengineered (modified or derived) from its native form to specificallyrecognize and induce a double-strand break in the desired recognitionsite. Thus, an engineered endonuclease can be derived from a native,naturally-occurring endonuclease or it could be artificially created orsynthesized. The modification of the endonuclease can be as little asone nucleotide. In some embodiments, the engineered endonuclease inducesa double-strand break in a recognition site, wherein the recognitionsite was not a sequence that would have been recognized by a native(non-engineered or non-modified) endonuclease. Producing a double-strandbreak in a recognition site or other DNA can be referred to herein as“cutting” or “cleaving” the recognition site or other DNA.

A “meganuclease” refers to a homing endonuclease, which like restrictionendonucleases, bind and cut at a specific recognition site, however therecognition sites for meganucleases are typically longer, about 18 bp ormore. In some embodiments of the invention, the meganuclease has beenengineered (or modified) to cut a specific endogenous recognitionsequence, wherein the endogenous target sequence prior to being cut bythe engineered double-strand-break-inducing agent was not a sequencethat would have been recognized by a native (non-engineered ornon-modified) endonuclease.

A “meganuclease polypeptide” refers to a polypeptide having meganucleaseactivity and thus capable of producing a double-strand break in therecognition sequence.

Meganucleases have been classified into four families based on conservedsequence motifs, the families are the LAGLIDADG, (SEQ ID NO: 437)GIYX_(n)YIG (wherein X is any amino acid and n can range from 10 to 11amino acids (for example SEQ ID NO: 438 for n =10), HX_(n)NX_(m)H(wherein X is any amino acid and n can range from 10 to 14 amino acidsand m can range from 7 to 8 amino acids; for example SEQ ID NO: 439 forn =10, m=8), and HXCX_(n)CXXXXHX_(m)C box families (wherein X is anyamino acid and n can range from 4 to 5 amino acids, m can range from 16to 17 amino acids; for example SEQ ID NO: 440 for n =4, m=16). (BelfortM, and Perlman P S J. Biol. Chem. 1995;270:30237-30240). These motifsparticipate in the coordination of metal ions and hydrolysis ofphosphodiester bonds. HEases are notable for their long recognitionsites, and for tolerating some sequence polymorphisms in their DNAsubstrates. The naming convention for meganuclease is similar to theconvection for other restriction endonuclease. Meganucleases are alsocharacterized by prefix F-,I-, or PI- for enzymes encoded byfree-standing open reading frames, introns, and inteins, respectively.For example, intron-, and freestanding gene encoded meganuclease fromSaccharomyces cerevisiae are denoted I-Scel, PI-Scel, and F-Scell,respectively. Meganuclease domains, structure and function are known,see for example, Guhan and Muniyappa (2003) Crit Rev Biochem Mol Biol38:199-248; Lucas et al., (2001) Nucleic Acids Res 29:960-9; Jurica andStoddard, (1999) Cell Mol Life Sci 55: 1304-26; Stoddard, (2006) Q RevBiophys 38:49-95; and Morue et al., (2002) Nat Struct Biol 9:764. Insome examples a naturally occurring variant, and/or engineeredderivative meganuclease is used. Methods for modifying the kinetics,cofactor interactions, expression, optimal conditions, and/orrecognition site specificity, and screening for activity are known, seefor example, Epinat et al., (2003) Nucleic Acids Res 31:2952-62;Chevalier et al., (2002) Mol Cell 10:895-905; Gimble et al., (2003) MolBiol 334:993-1008; Seligman et al., (2002) Nucleic Acids Res 30:3870-9;Sussman et al., (2004) J Mol Biol 342:31-41; Rosen et al., (2006)Nucleic Acids Res 34:4791-800; Chames et al., (2005) Nucleic Acids Res33:e178; Smith et al., (2006) Nucleic Acids Res 34:e149; Gruen et al.,(2002) Nucleic Acids Res 30e29; Chen and Zhao, (2005) Nucleic Acids Res33:e154; WO2005105989; WO2003078619; WO2006097854; WO2006097853;WO2006097784; and WO2004031346.

Any meganuclease can be used herein, including, but not limited to,I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-CeuI,I-CeuAIIP, I-CreI, I-CrepsbIP, I-CrepsbIIIP, I-CrepsbIIIP, I-CrepsbIVP,I-TliI, I-PpoI, PI-PspI, F-SceI, F-SceII, F-SuvI, F-TevI, F-TevII,I-AmaI, I-Anil, I-ChuI, I-CmoeI, I-CpaI, I-CpaII, I-CsmI, I-CvuI,I-CvuAIP, I-DdiI, I-DdiII, I-DirI, I-DmoI, I-HmuI, I-HmuII, I-HsNIP,I-LlaI, I-MsoI, I-NaaI, I-NanI, I-NcIIP, I-NgrIP, I-NitI, I-NjaI,I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP,I-PobIP, I-PorI, I-PorIIP, I-PbpIP, I-SpBetaIP, I-ScaI, I-SexIP,I-SneIP, I-SpomI, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp6803I,I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, I-TdeIP, I-TevI, I-TevII,I-TevIII, I-UarAP, I-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MtuI,PI-MtuHIP PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII,PI-Rma43812IP, PI-SpBetaIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI,PI-TliI, PI-TliII, or any active variants or fragments thereof. In aspecific embodiment, the engineered endonuclease is derived from I-Cre-Ihaving the sequence set forth in SEQ ID NO: 15, 21 or 26 or an activevariant or fragment thereof.

TAL effector nucleases are a new class of sequence-specific nucleasesthat can be used to make double-strand breaks at specific targetsequences in the genome of a plant or other organism. TAL effectornucleases are created by fusing a native or engineered transcriptionactivator-like (TAL) effector, or functional part thereof, to thecatalytic domain of an endonuclease, such as, for example, FokI. Theunique, modular TAL effector DNA binding domain allows for the design ofproteins with potentially any given DNA recognition specificity. Thus,the DNA binding domains of the TAL effector nucleases can be engineeredto recognize specific DNA target sites and thus, used to makedouble-strand breaks at desired target sequences. See, WO 2010/079430;Morbitzer et al. (2010) PNAS 10.1073/pnas.1013133107; Scholze & Boch(2010) Virulence 1:428-432; Christian et al. Genetics (2010)186:757-761; Li et al. (2010) Nuc. Acids Res. (2010)doi:10.1093/nar/gkq704; and Miller et al. (2011) Nature Biotechnology29:143-148; all of which are herein incorporated by reference.

The term “meganuclease activity” as used herein refers to the ability ofa meganuclease to cut at a desired recognition sequence and thus retaindouble-strand-break-inducing activity.

Assays for meganuclease activity are known and generally measure theoverall activity and specificity of the meganuclease on DNA substratescontaining the recognition site. For example, the meganuclease activitycan be measured using a yeast screening assay as described herein. Yeastcells with a functional Ade2 gene are white, whereas those lacking Ade2function exhibit red pigmentation due to accumulation of a metaboliteearlier in the adenine biosynthetic pathway resulting in red colonieswith white sectors as shown in FIGS. 2 and 3. The degree of whitesectoring, sometimes extending to entire colonies, indicates the amountof meganuclease cutting activity. Since the sectoring phenotype is aqualitative measure of meganuclease activity, a 0-4 numerical scoringsystem was implemented. As shown in FIG. 3, a score of 0 indicates thatno white sectors (no meganuclease cutting) were observed; a score of 4indicates completely white colonies (complete cutting of the recognitionsite); scores of 1-3 indicate intermediate white sectoring phenotypes(and intermediate degrees of recognition site cutting). Meganucleaseactivity can also be measured in-vitro as described herein. In short,time-course digestions can be carried out on plasmid DNA containing themeganuclease recognition site at 37° C., 28° C., and 23° C. and the %digestion of each sample or loss of meganuclease recognition sites(indicative of meganuclease activity) can be determined by real-timePCR. Furthermore, meganuclease activity can be measured in-planta bydetermining the Target Site (TS) mutation rate. Target site mutationrate is defined as: (number of events with target sitemodification/total number events)*100%.

An “increased” or an “increased” activity are used interchangeablyherein. An “increased” or “increased” meganuclease activity comprisesany statistically significant increase in the activity of the parentalmeganuclease polypeptide as determined through any activity assaysdescribed herein.

The meganuclease can be provided via a polynucleotide encoding theendonuclease. Such a polynucleotide encoding an endonuclease can bemodified to substitute codons having a higher frequency of usage in aplant, as compared to the naturally occurring polynucleotide sequence.For example the polynucleotide encoding the meganuclease can be modifiedto substitute codons having a higher frequency of usage in a maize orsoybean plant, as compared to the naturally occurring polynucleotidesequence.

Various methods and compositions are provided which employpolynucleotides and polypeptides having meganuclease activity.

In one embodiment, the invention concerns an isolated or recombinantpolynucleotide comprising a nucleotide sequence encoding a meganucleasepolypeptide, said polypeptide comprising: a) an amino acid sequencehaving at least one amino acid modification at an amino acid positioncorresponding to a position of SEQ ID NO: 1 selected from the groupconsisting of positions 2, 12, 16, 22, 23, 31, 36, 43, 50, 56, 58, 59,62, 71, 72, 73, 80, 81, 82, 86, 91, 95, 98, 103, 113, 114, 116, 117,118, 121, 124, 128, 129, 131, 147, 151, 153, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,194, 195, 196, 197, 200, 203, 204, 209, 222, 232, 236, 237, 246, 254,258, 267, 278, 281, 282, 289, 308, 311, 312, 316, 318, 319, 334, 339,340, 342, 345, 346, 348 and combinations thereof; or, b) an amino acidsequence having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44 of any of the aminoacid modification of (a).

In another embodiment, the invention concerns an isolated or recombinantpolynucleotide of the current disclosure, and its correspondingpolypeptide, wherein said nucleotide sequence encodes a meganucleasepolypeptide, wherein said polypeptide further comprises at least oneamino acid modification described herein such as those shown in FIG.5A-FIG. 5E, FIG. 9A-FIG. 9N, FIG. 10A-FIG. 10D, FIG. 11, FIG. 12, FIG.13, FIG. 14A-FIG. 14F and FIG. 15A-FIG. 15E as well any I-Cre1 typemodification known and any combination thereof.

Further provided are methods and compositions which employpolynucleotides and polypeptides having increased meganuclease activitywhen compared to an appropriate control. Such meganuclease polypeptidesinclude those set forth in any one of SEQ ID NOs:14-38 (LIG3-4variants), SEQ ID NOs:87-167 (MHP77 variants, SEQ ID NOs: 251.252, 253,262 (MHP77.3 variants), SEQ ID NOs:272-275 (MS26+ variants), SEQ IDNOs:284-298 (MHP14 variants), SEQ ID NOs:315-320 (MHP14+ variants), SEQID NOs: 330-341 (MH107 variants), SEQ ID NOs:357-371 (ZM6.3 variants),SEQ ID NOs:390-403 (ZM6.22V2 variants) or SEQ ID NOs: 430-432 andbiologically active variants thereof. Further provided are thepolynucleotides encoding these various polypeptides and active variantthereof.

The term “Variant” protein is intended to mean a protein derived fromthe protein (referred to as parental protein) by deletion (i.e.,truncation at the 5′ and/or 3′ end) and/or a deletion or addition of oneor more amino acids at one or more internal sites in the parentalprotein and/or substitution of one or more amino acids at one or moresites in the parental protein. As used herein, a “parental”polynucleotide, polypeptide (protein) can result from human manipulationor from a native protein comprising a naturally occurring nucleotidesequence or amino acid sequence, respectively. Variant proteinsencompassed are biologically active, that is they continue to possessthe desired biological activity of the parental protein, that is, havemeganuclease activity. Such variants may result from, for example,genetic polymorphism or from human manipulation.

The term “variant meganuclease” refers to a variant protein withmeganuclease activity. The variant meganuclease is derived from aparental meganuclease wherein the variant meganuclease comprises atleast one amino acid modification when compared to the parentalmeganuclease polypeptide.

Variant meganuclease polypeptides of the invention include those setforth in any one of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 251, 252, 253, 262, 272, 273, 274, 275,284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,298, 315, 316, 317, 318, 319, 320, 330, 331, 332, 334, 335, 336, 337,338, 339, 340, 341, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 370, 371, 390, 391, 392, 393, 394, 395, 396, 397,398, 399, 400, 401, 402, 403, 430, 431, 432 or 433 and biologicallyactive variants and fragments thereof. Further provided are thepolynucleotides encoding these various polypeptides and active variantand fragments thereof.

Any one of the amino acid modifications identified in Examples 3-23 canbe transferred to a parental meganuclease to create a variantmeganuclease. These meganucleases can be screened for increased activityby methods described herein.

One embodiment of the invention concerns the transfer of at least oneamino acid modification selected from the group of Y12 to H, G19 to S orA, Q50 to K or R, F54 to I, D56 to L, V105 to A, E124 to R, V129 to A,I132 to V or T, D153 to M or L, V316 to A or I 319 to V to a parentalmeganuclease in order to improve the activity of the parentalmeganuclease. Another embodiment concern the transfer of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, or 12 amino acid modification selected from thegroup of Y12 to H, G19 to S or A, Q50 to K or R, F54 to I, D56 to L,V105 to A, E124 to R, V129 to A, I132 to V or T, D153 to M or L, V316 toA or I 319 to V to a parental meganuclease in order to improve theactivity of the parental meganuclease.

Any one of the modifications described herein can be combined with otherknown modifications of I-CreI type meganucleases.

As used herein with respect to a recombinant polynucleotide encoding arecombinant protein, term “modification” means any insertion, deletionor substitution of an amino acid residue in the recombinant proteinsequence relative to a reference or control sequence.

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the meganuclease polypeptides of the invention.Naturally occurring variants such as these can be identified with theuse of well-known molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant polynucleotides also include synthetically derivedpolynucleotides, such as those generated, for example, by usingsite-directed mutagenesis or gene synthesis but which still encode ameganuclease polypeptide.

Biologically Active variants of meganucleases (i.e. variantmeganucleases) are also provided. Variant meganucleases are biologicallyactive variants of a meganuclease polypeptide (and the polynucleotideencoding the same) will have at least about 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 95.7%,95.9%, 96%, 96.3%, 96.5%, 96.9%, 97%, 97.3%, 97.5%, 97.9%, 98%, 98.3%,98.5%, 98.9%, 99%, 99.3%, 99.5%, 99.6% or more sequence identity to thepolypeptide of a control meganuclease, wherein the active variantsretain the ability to cut at a desired recognition site. For example,any of the variant meganucleases described herein can be modified from aparental endonuclease sequence and designed to recognize and induce adouble strand break at the same recognition site of the parentalmeganuclease. Thus in some embodiments, the variant meganucleasecontains at least one amino acid modification when compared to theparental meganuclease and has a specificity to induce a double-strandbreak at the same recognition sequence as the corresponding parentalmeganuclease recognition sequence.

A “control meganuclease” or “reference meganuclease” can be usedinterchangeably and refers to any meganuclease to which a variantmeganuclease is compared to. Control meganucleases can include, but arenot limited to, parental or corresponding meganucleases or any wild-typeI-Cre1 type meganucleases.

Numbering of an amino acid or nucleotide polymer, such any one of themeganucleases of the invention, corresponds to numbering of a selectedamino acid polymer or nucleic acid when the position of a given monomercomponent (amino acid residue, incorporated nucleotide, etc.) of thepolymer corresponds to the same residue position in a selected referencepolypeptide or polynucleotide.

Further provided are biologically active variants of a meganucleasepolypeptide (and the polynucleotide encoding the same) that will have atleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 95.5%, 95.7%, 95.9%, 96%, 96.3%, 96.5%, 96.9%, 97%,97.3%, 97.5%, 97.9%, 98%, 98.3%, 98.5%, 98.9%, 99%, 99.3%, 99.5%, 99.6%or more sequence identity to the polypeptide of any one of SEQ ID NO:14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 251,252, 253, 262, 272, 273, 274, 275, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 315, 316, 317, 318, 319, 320,330, 331, 332, 334, 335, 336, 337, 338, 339, 340, 341, 357, 358, 359,360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 370, 371, 390,391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 430,431, 432 or 433 or with regard to any of the meganuclease polypeptidesdisclosed herein as determined by sequence alignment

In one embodiment the variant meganuclease of the present inventioncomprises a linker polypeptide, wherein said linker polypeptidecomprises: a) SEQ ID NO: 420; b) SEQ ID NO: 421; c) SEQ ID NO: 422; or,d) an amino acid sequence consisting of any possible amino acid atpositions corresponding to positions 156 to 193 of SEQ ID NO: 1. It isalso understood that these linker sequences can be substituted for anyother linker sequence that links both I-Cre type monomers while stillenabling the single polypeptide meganuclease to provide a double strandbreak at a target sequence.

As used herein, a “genomic region of interest” is a segment of achromosome in the genome of a plant that is desirable for introducing apolynucleotide of interest or trait of interest. The genomic region ofinterest can include, for example, one or more polynucleotides ofinterest. Generally, a genomic region of interest of the presentinvention comprises a segment of chromosome that is 0-15 cM.

As used herein, a “polynucleotide of interest” within a genomic regionof interest is any coding and/or non-coding portion of the genomicregion of interest including, but not limited to, a transgene, a nativegene, a mutated gene, and a genetic marker such as, for example, asingle nucleotide polymorphism (SNP) marker and a simple sequence repeat(SSR) marker.

As used herein, “physically linked,” “in physical linkage”, and“genetically linked” are used to refer to any two or more genes,transgenes, native genes, mutated genes, alterations, target sites,markers, and the like that are part of the same DNA molecule orchromosome.

Sequence Comparisons

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides or polypeptides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, and, (d)“percent sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or gene sequenceor protein sequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polypeptide sequence, wherein the polypeptidesequence in the comparison window may comprise additions or deletions(i.e., gaps) compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two polypeptides.Generally, the comparison window is at least 5, 10, 15, or 20 contiguousamino acid in length, or it can be 30, 40, 50, 100, or longer. Those ofskill in the art understand that to avoid a high similarity to areference sequence due to inclusion of gaps in the polypeptide sequencea gap penalty is typically introduced and is subtracted from the numberof matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Computerimplementations of these mathematical algorithms can be utilized forcomparison of sequences to determine sequence identity. Suchimplementations include, but are not limited to: CLUSTAL in the PC/Geneprogram (available from Intelligenetics, Mountain View, Calif.); theALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTAin the GCG Wisconsin Genetics Software Package, Version 10 (availablefrom Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA).Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. BLASTP protein searches can beperformed using default parameters. See,blast.ncbi.nlm.nih.gov/Blast.cgi.

Sequence alignments and percent similarity calculations may bedetermined using the Megalign program of the LASARGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.) or using the AlignXprogram of the Vector NTI bioinformatics computing suite (Invitrogen,Carlsbad, Calif.). Multiple alignment of the sequences are performedusing the Clustal method of alignment (Higgins and Sharp, CABIOS5:151-153 (1989)) with the default parameters (GAP PENALTY=10, GAPLENGTH PENALTY=10). Default parameters for pairwise alignments andcalculation of percent identity of protein sequences using the Clustalmethod are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Fornucleic acids these parameters are GAP PENALTY=10, GAP LENGTHPENALTY=10, KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. A“substantial portion” of an amino acid or nucleotide sequence comprisesenough of the amino acid sequence of a polypeptide or the nucleotidesequence of a gene to afford putative identification of that polypeptideor gene, either by manual evaluation of the sequence by one skilled inthe art, or by computer-automated sequence comparison and identificationusing algorithms such as BLAST (Altschul, S. F. et al., J. Mol. Biol.215:403-410 (1993)) and Gapped Blast (Altschul, S. F. et al., NucleicAcids Res. 25:3389-3402 (1997)). BLASTN refers to a BLAST program thatcompares a nucleotide query sequence against a nucleotide sequencedatabase.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” or “recombinant expressionconstruct”, which are used interchangeably, refers to any gene that isnot a native gene, comprising regulatory and coding sequences that arenot found together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a DNA sequence which codes for a specificamino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may include, butare not limited to, promoters, translation leader sequences, introns,and polyadenylation recognition sequences.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for increased expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

As used herein, “sequence identity” or “identity” in the context of twopolynucleotides or polypeptide sequences makes reference to the residuesin the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity). When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percent sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

As used herein, “percent sequence identity” means the value determinedby comparing two aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison, and multiplyingthe result by 100 to yield the percent sequence identity.

Polynucleotide Constructs

Provided herein are polynucleotides or nucleic acid molecules comprisingthe meganucleases or any active variants or fragments thereof. The terms“polynucleotide,” “polynucleotide sequence,” “nucleic acid sequence,”and “nucleic acid fragment” are used interchangeably herein. These termsencompass nucleotide sequences and the like. The use of the term“polynucleotide” is not intended to limit the present invention topolynucleotides comprising DNA. Those of ordinary skill in the art willrecognize that polynucleotides can comprise ribonucleotides andcombinations of ribonucleotides and deoxyribonucleotides. Suchdeoxyribonucleotides and ribonucleotides include both naturallyoccurring molecules and synthetic analogues. The polynucleotides of theinvention also encompass all forms of sequences including, but notlimited to, single-stranded forms, double-stranded forms, hairpins,stem-and-loop structures, and the like.

Further provided are recombinant polynucleotides comprising the variousmeganucleases. The terms “recombinant polynucleotide”, “recombinantnucleotide”, “recombinant DNA” and “recombinant DNA construct” are usedinterchangeably herein. A recombinant construct comprises an artificialor heterologous combination of nucleic acid sequences, e.g., regulatoryand coding sequences that are not found together in nature. For example,a transfer cassette can comprise restriction sites and a heterologouspolynucleotide of interest. In other embodiments, a recombinantconstruct may comprise regulatory sequences and coding sequences thatare derived from different sources, or regulatory sequences and codingsequences derived from the same source, but arranged in a mannerdifferent than that found in nature. Such a construct may be used byitself or may be used in conjunction with a vector. If a vector is used,then the choice of vector is dependent upon the method that will be usedto transform host cells as is well known to those skilled in the art.For example, a plasmid vector can be used. The skilled artisan is wellaware of the genetic elements that must be present on the vector inorder to successfully transform, select and propagate host cellscomprising any of the isolated nucleic acid fragments provided herein.The skilled artisan will also recognize that different independenttransformation events will result in different levels and patterns ofexpression (Jones et al., EMBO J. 4:2411-2418 (1985); De Almeida et al.,Mol. Gen. Genetics 218:78-86 (1989)), and thus that multiple events mustbe screened in order to obtain lines displaying the desired expressionlevel and pattern. Such screening may be accomplished by Southernanalysis of DNA, Northern analysis of mRNA expression, immunoblottinganalysis of protein expression, or phenotypic analysis, among others.

The meganuclease polynucleotides disclosed herein can be provided inexpression cassettes for expression in the plant of interest. Thecassette can include 5′ and 3′ regulatory sequences operably linked toan meganuclease polynucleotide or active variant or fragment thereof.“Operably linked” is intended to mean a functional linkage between twoor more elements. For example, an operable linkage between apolynucleotide of interest and a regulatory sequence (i.e., a promoter)is a functional link that allows for expression of the polynucleotide ofinterest. Operably linked elements may be contiguous or non-contiguous.When used to refer to the joining of two protein coding regions, byoperably linked is intended that the coding regions are in the samereading frame. The cassette may additionally contain at least oneadditional gene to be cotransformed into the organism. Alternatively,the additional gene(s) can be provided on multiple expression cassettes.Such an expression cassette is provided with a plurality of restrictionsites and/or recombination sites for insertion of the meganucleasepolynucleotide or active variant or fragment thereof to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

The expression cassette can include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a meganuclease polynucleotide or active variant orfragment thereof, and a transcriptional and translational terminationregion (i.e., termination region) functional in plants. The regulatoryregions (i.e., promoters, transcriptional regulatory regions, andtranslational termination regions) and/or the meganucleasepolynucleotide or active variant or fragment thereof may benative/analogous to the host cell or to each other. Alternatively, theregulatory regions and/or the meganuclease polynucleotide of or activevariant or fragment thereof may be heterologous to the host cell or toeach other.

As used herein, “heterologous” in reference to a sequence is a sequencethat originates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived,or, if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus, or the promoteris not the native promoter for the operably linked polynucleotide.

While it may be optimal to express the sequences using heterologouspromoters, the native promoter sequences may be used. Such constructscan change expression levels of the meganuclease polynucleotide in theplant or plant cell. Thus, the phenotype of the plant or plant cell canbe altered.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked meganucleasepolynucleotide or active variant or fragment thereof, may be native withthe plant host, or may be derived from another source (i.e., foreign orheterologous) to the promoter, the meganuclease polynucleotide or activefragment or variant thereof, the plant host, or any combination thereof.Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also Guerineau et al. (1991) Mol. Gen. Genet.262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991)Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroeet al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

Where appropriate, the polynucleotides may be optimized for increasedexpression in the transformed plant. That is, the polynucleotides can besynthesized using plant-preferred codons for improved expression. See,for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallieet al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus) (Virology 154:9-20), and human immunoglobulin heavy-chain bindingprotein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslatedleader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4)(Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader(TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss,New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV)(Lommel et al. (1991) Virology 81:382-385. See also, Della-Cioppa et al.(1987) Plant Physiol. 84:965-968.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used to express the various meganucleasesequence disclosed herein, including the native promoter of thepolynucleotide sequence of interest. The promoters can be selected basedon the desired outcome. Such promoters include, for example,constitutive, tissue-preferred, or other promoters for expression inplants.

Constitutive promoters include, for example, the core promoter of theRsyn7 promoter and other constitutive promoters disclosed in WO 99/43838and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al.(1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689);pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten etal. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026),and the like. Other constitutive promoters include, for example, U.S.Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Tissue-preferred promoters can be utilized to target enhancedmeganuclease expression within a particular plant tissue.Tissue-preferred promoters include those described in Yamamoto et al.(1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant CellPhysiol. 38(7):792-803; Hansen et al. (1997)Mol. Gen Genet.254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168;Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al.(1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) PlantPhysiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozcoet al. (1993) Plant Mol Biol. 23(6): 1129-1138; Matsuoka et al. (1993)Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al.(1993) Plant J. 4(3):495-505. Such promoters can be modified, ifnecessary, for weak expression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6): 1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Synthetic promoters can be used to express meganuclease sequences orbiologically active variants and fragments thereof

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglyphosate, glufosinate ammonium, bromoxynil, sulfonylureas, dicamba,and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su et al. (2004)Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. CellScience 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), andyellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al.(2004) J. Cell Science 117:943-54). For additional selectable markers,see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318;Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge etal. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad.Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl.Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference. Theabove list of selectable marker genes is not meant to be limiting. Anyselectable marker gene can be used in the present invention.

Method of Introducing

The meganuclease may be introduced by any means known in the art. Forexample, a cell, yeast or plant having the recognition site in itsgenome is provided. The meganuclease may be transiently expressed or thepolypeptide itself can be directly provided to the cell. Alternatively,a nucleotide sequence capable of expressing the meganuclease may bestably integrated into the genome of the plant. In the presence of thecorresponding recognition site and the meganuclease, a donor DNA can beinserted into the transformed plant's genome. Alternatively, thedifferent components may be brought together by sexually crossingtransformed plants. Thus a sequence encoding a meganuclease and/ortarget site can be sexually crossed to one another to allow eachcomponent of the system to be present in a single plant. Themeganuclease may be under the control of a constitutive or induciblepromoter. Such promoters of interest are discussed in further detailelsewhere herein.

Various methods can be used to introduce a sequence of interest such as,any of the meganuclease of the invention, into a plant or plant part.“Introducing” is intended to mean presenting to the plant, plant cell orplant part the polynucleotide or polypeptide in such a manner that thesequence gains access to the interior of a cell of the plant. Themethods of the invention do not depend on a particular method forintroducing a sequence into a plant or plant part, only that thepolynucleotide or polypeptides gains access to the interior of at leastone cell of the plant. Methods for introducing polynucleotide orpolypeptides into plants are known in the art including, but not limitedto, stable transformation methods, transient transformation methods, andvirus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” is intended to mean that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway et al.(1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediatedtransformation (U.S. Pat. No. 5,563,055 and U.S. Pat. No. 5,981,840),direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), andballistic particle acceleration (see, for example, U.S. Pat. No.4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. No. 5,886,244; and, U.S.Pat. No. 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and OrganCulture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag,Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Lec1transformation (WO 00/28058). Also see Weissinger et at. (1988) Ann.Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science andTechnology 5:27-37 (onion); Christou et al. (1988) Plant Physiol.87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926(soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat.Nos. 5,240,855; 5,322,783; and, 5,324,646; Klein et al. (1988) PlantPhysiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987)Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al.(1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman etal. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990)Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl.Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al.(1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) PlantCell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

In specific embodiments, the meganuclease sequences or active variant orfragments thereof can be provided to a yeast cell or plant using avariety of transient transformation methods. Such transienttransformation methods include, but are not limited to, the introductionof the meganuclease protein or active variants and fragments thereofdirectly into a yeast cell or plant. Such methods include, for example,microinjection or particle bombardment. See, for example, Crossway etal. (1986) Mol Gen. Genet. 202:179-185; Nomura et al. (1986) Plant Sci.44:53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 andHush et al. (1994) The Journal of Cell Science 107:775-784, all of whichare herein incorporated by reference.

In other embodiments, the polynucleotide of the invention may beintroduced into yeast cells or plants by contacting plants with a virusor viral nucleic acids. Generally, such methods involve incorporating anucleotide construct of the invention within a DNA or RNA molecule. Itis recognized that the an meganuclease sequence may be initiallysynthesized as part of a viral polyprotein, which later may be processedby proteolysis in vivo or in vitro to produce the desired recombinantprotein. Further, it is recognized that promoters of the invention alsoencompass promoters utilized for transcription by viral RNA polymerases.Methods for introducing polynucleotides into plants and expressing aprotein encoded therein, involving viral DNA or RNA molecules, are knownin the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190,5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) MolecularBiotechnology 5:209-221; herein incorporated by reference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide of the invention can be contained in transfercassette flanked by two non-recombinogenic recombination sites. Thetransfer cassette is introduced into a plant having stably incorporatedinto its genome a target site which is flanked by two non-recombinogenicrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The polynucleotide ofinterest is thereby integrated at a specific chromosomal position in theplant genome. Other methods to target polynucleotides are set forth inWO 2009/114321 (herein incorporated by reference), which describes“custom” meganucleases produced to modify plant genomes, in particularthe genome of maize. See, also, Gao et al. (2010) Plant Journal1:176-187.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting progeny having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a polynucleotide of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

Method of Detections

Methods for detecting a meganuclease polypeptide or an active variant orfragment thereof are provided. Such methods comprise analyzing planttissues to detect such polypeptides or the polynucleotides encoding thesame. The detection methods can directly assay for the presence of themeganuclease polypeptide or polynucleotide or the detection methods canindirectly assay for the sequences by assaying the phenotype of thecell, yeast, plant, plant cell or plant explant expressing the sequence.

In still other embodiments, the meganuclease polypeptide or activevariant or fragment thereof can be detected in a plant tissue bydetecting the presence of a polynucleotide encoding any of the variousmeganuclease polypeptides or active variants and fragments thereof. Inone embodiment, the detection method comprises assaying plant tissueusing PCR amplification.

As used herein, “primers” are isolated polynucleotides that are annealedto a complementary target DNA strand by nucleic acid hybridization toform a hybrid between the primer and the target DNA strand, thenextended along the target DNA strand by a polymerase, e.g., a DNApolymerase. Primer pairs of the invention refer to their use foramplification of a target polynucleotide, e.g., by the polymerase chainreaction (PCR) or other conventional nucleic-acid amplification methods.“PCR” or “polymerase chain reaction” is a technique used for theamplification of specific DNA segments (see, U.S. Pat. Nos. 4,683,195and 4,800,159; herein incorporated by reference).

Probes and primers are of sufficient nucleotide length to bind to thetarget DNA sequence and specifically detect and/or identify apolynucleotide encoding a meganuclease polypeptide or active variant orfragment thereof as describe elsewhere herein. It is recognized that thehybridization conditions or reaction conditions can be determined by theoperator to achieve this result. This length may be of any length thatis of sufficient length to be useful in a detection method of choice.Such probes and primers can hybridize specifically to a target sequenceunder high stringency hybridization conditions. Probes and primersaccording to embodiments of the present invention may have complete DNAsequence identity of contiguous nucleotides with the target sequence,although probes differing from the target DNA sequence and that retainthe ability to specifically detect and/or identify a target DNA sequencemay be designed by conventional methods. Accordingly, probes and primerscan share about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or greater sequence identity or complementarity to the targetpolynucleotide.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2.sup.nd ed, vol.1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. 1989 (hereinafter, “Sambrook et al., 1989”); CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates)(hereinafter, “Ausubel et al., 1992”); and Innis et al., PCR Protocols:A Guide to Methods and Applications, Academic Press: San Diego, 1990.PCR primer pairs can be derived from a known sequence, for example, byusing computer programs intended for that purpose such as the PCR primeranalysis tool in Vector NTI version 10 (Invitrogen); PrimerSelect(DNASTAR Inc., Madison, Wis.); and Primer (Version 0.5.COPYRGT., 1991,Whitehead Institute for Biomedical Research, Cambridge, Mass.).Additionally, the sequence can be visually scanned and primers manuallyidentified using guidelines known to one of skill in the art.

Method of Identifying Meganuclease Variants.

Various methods can be employed to identify further meganucleasevariants. The polynucleotides of the invention are optionally used assubstrates for a variety of diversity generating procedures, e.g.,mutation, recombination and recursive recombination reactions, inaddition to their use in standard cloning methods as set forth in, e.g.,Ausubel, Berger and Sambrook, i.e., to produce additional meganucleasepolynucleotides and polypeptides with desired properties. A variety ofdiversity generating protocols can be used. The procedures can be usedseparately, and/or in combination to produce one or more variants of apolynucleotide or set of polynucleotides, as well variants of encodedproteins. Individually and collectively, these procedures providerobust, widely applicable ways of generating diversified polynucleotidesand sets of polynucleotides (including, e.g., polynucleotide libraries)useful, e.g., for the engineering or rapid evolution of polynucleotides,proteins, pathways, cells and/or organisms with new and/or improvedcharacteristics. The process of altering the sequence can result in, forexample, single nucleotide substitutions, multiple nucleotidesubstitutions, and insertion or deletion of regions of the nucleic acidsequence.

While distinctions and classifications are made in the course of theensuing discussion for clarity, it will be appreciated that thetechniques are often not mutually exclusive. Indeed, the various methodscan be used singly or in combination, in parallel or in series, toaccess diverse sequence variants.

The terms “diversification” and “diversity,” as applied to apolynucleotide, refers to generation of a plurality of modified forms ofa parental polynucleotide, or plurality of parental polynucleotides. Inthe case where the polynucleotide encodes a polypeptide, diversity inthe nucleotide sequence of the polynucleotide can result in diversity inthe corresponding encoded polypeptide, e.g. a diverse pool ofpolynucleotides encoding a plurality of polypeptide variants. In someembodiments of the invention, this sequence diversity is exploited byscreening/selecting a library of diversified polynucleotides forvariants with desirable functional attributes, e.g., a polynucleotideencoding a meganuclease with enhanced functional characteristics.

The result of any of the diversity generating procedures describedherein can be the generation of one or more polynucleotides, which canbe selected or screened for polynucleotides that encode proteins with orwhich confer desirable properties. Following diversification by one ormore of the methods herein, or otherwise available to one of skill, anypolynucleotides that are produced can be selected for a desired activityor property, e.g. altered Km, use of alternative cofactors, increasedkcat, etc. This can include identifying any activity that can bedetected, for example, in an automated or automatable format, by any ofthe assays in the art. For example, modified meganuclease polypeptidescan be detected by assaying for a meganuclease activity. Assays tomeasure such activity are described elsewhere herein. A variety ofrelated (or even unrelated) properties can be evaluated, in serial or inparallel, at the discretion of the practitioner.

Descriptions of a variety of diversity generating procedures, includingfamily shuffling and methods for generating modified nucleic acidsequences encoding multiple enzymatic domains, are found in thefollowing publications and the references cited therein: Soong N. et al.(2000) Nat Genet 25(4):436-39; Stemmer et al. (1999) Tumor Targeting4:1-4; Ness et al. (1999) Nature Biotechnology 17:893-896; Chang et al.(1999) Nature Biotechnology 17:793-797; Minshull and Stemmer (1999)Current Opinion in Chemical Biology 3:284-290; Christians et al. (1999)Nature Biotechnology 17:259-264; Crameri et al. (1998) Nature391:288-291; Crameri et al. (1997) Nature Biotechnology 15:436-438;Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Patten etal. (1997) Current Opinion in Biotechnology 8:724-733; Crameri et al.(1996) Nature Medicine 2:100-103; Crameri et al. (1996) NatureBiotechnology 14:315-319; Gates et al. (1996) Journal of MolecularBiology 255:373-386; Stemmer (1996) “Sexual PCR and Assembly PCR” In:The Encyclopedia of Molecular Biology. VCH Publishers, New York. pp.447-457; Crameri and Stemmer (1995) BioTechniques 18:194-195; Stemmer etal. (1995) Gene: 164:49-53; Stemmer (1995) Science 270: 1510; Stemmer(1995) Bio/Technology 13:549-553; Stemmer (1994) Nature 370:389-391; andStemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. See alsoWO2008/073877 and US 20070204369, both of which are herein incorporatedby reference in their entirety.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) Anal Biochem. 254(2):157-178; Dale et al. (1996) Methods Mol. Biol. 57:369-374; Smith (1985)Ann. Rev. Genet. 19:423-462; Botstein & Shortle (1985) Science229:1193-1201; Carter (1986) Biochem. J. 237:1-7; and Kunkel (1987)Nucleic Acids & Molecular Biology (Eckstein, F. and Lilley, D. M. J.eds., Springer Verlag, Berlin)); mutagenesis using uracil containingtemplates (Kunkel (1985) Proc. Natl. Acad Sci. USA 82:488-492; Kunkel etal. (1987) Methods in Enzymol. 154, 367-382; and Bass et al. (1988)Science 242:240-245); oligonucleotide-directed mutagenesis (Methods inEnzymol. 100: 468-500 (1983); Methods in Enzymol. 154: 329-350 (1987);Zoller & Smith (1982) Nucleic Acids Res. 10:6487-6500; Zoller & Smith(1983) Methods in Enzymol. 100:468-500; and Zoller & Smith (1987)Methods in Enzymol. 154:329-350); phosphorothioate-modified DNAmutagenesis (Taylor et al. (1985) Nucl. Acids Res. 13: 8749-8764; Tayloret al. (1985) Nucl. Acids Res. 13: 8765-8787 (1985); Nakamaye & Eckstein(1986) Nucl. Acids Res. 14: 9679-9698; Sayers et al. (1988) Nucl. AcidsRes. 16:791-802; and Sayers et al. (1988) Nucl. Acids Res. 16: 803-814);mutagenesis using gapped duplex DNA (Kramer et al. (1984) Nucl. AcidsRes. 12: 9441-9456; Kramer & Fritz (1987) Methods in Enzymol.154:350-367; Kramer et al. (1988) Nucl. Acids Res. 16: 7207; and Fritzet al. (1988) Nucl. Acids Res. 16: 6987-6999).

Additional suitable methods include, but are not limited to, pointmismatch repair (Kramer et al. (1984) Cell 38:879-887), mutagenesisusing repair-deficient host strains (Carter et al. (1985) Nucl. AcidsRes. 13: 4431-4443; and Carter (1987) Methods in Enzymol. 154: 382-403),deletion mutagenesis (Eghtedarzadeh & Henikoff (1986) Nucl. Acids Res.14: 5115), restriction-selection and restriction-purification (Wells etal. (1986) Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis bytotal gene synthesis (Nambiar et al. (1984) Science 223: 1299-1301;Sakamar and Khorana (1988) Nucl. Acids Res. 14: 6361-6372; Wells et al.(1985) Gene 34:315-323; and Grundström et al. (1985) Nucl. Acids Res.13: 3305-3316), and double-strand break repair (Mandecki (1986); Arnold(1993) Current Opinion in Biotechnology 4:450-455 and Proc. Natl. Acad.Sci. USA, 83:7177-7181). Additional details on many of the above methodscan be found in Methods in Enzymology Volume 154, which also describesuseful controls for trouble-shooting problems with various mutagenesismethods.

Additional details regarding various diversity generating methods can befound in the following U.S. patents, PCT publications, and EPOpublications: U.S. Pat. No. 5,605,793, U.S. Pat. No. 5,811,238, U.S.Pat. No. 5,830,721, U.S. Pat. No. 5,834,252, U.S. Pat. No. 5,837,458, WO95/22625, WO 96/33207, WO 97/20078, WO 97/35966, WO 99/41402, WO99/41383, WO 99/41369, WO 99/41368, EP 752008, EP 0932670, WO 99/23107,WO 99/21979, WO 98/31837, WO 98/27230, WO 98/13487, WO 00/00632, WO00/09679, WO 98/42832, WO 99/29902, WO 98/41653, WO 98/41622, WO98/42727, WO 00/18906, WO 00/04190, WO 00/42561, WO 00/42559, WO00/42560, WO 01/23401, and, PCT/US01/06775. See, also WO20074303, hereinincorporated by reference.

In brief, several different general classes of sequence modificationmethods, such as mutation, recombination, etc. are applicable to thepresent invention and set forth, e.g., in the references above. That is,alterations to the component nucleic acid sequences to produced modifiedgene fusion constructs can be performed by any number of the protocolsdescribed, either before cojoining of the sequences, or after thecojoining step. The following exemplify some of the different types ofpreferred formats for diversity generation in the context of the presentinvention, including, e.g., certain recombination based diversitygeneration formats.

Nucleic acids can be recombined in vitro by any of a variety oftechniques discussed in the references above, including e.g., DNAsedigestion of nucleic acids to be recombined followed by ligation and/orPCR reassembly of the nucleic acids. For example, sexual PCR mutagenesiscan be used in which random (or pseudo random, or even non-random)fragmentation of the DNA molecule is followed by recombination, based onsequence similarity, between DNA molecules with different but relatedDNA sequences, in vitro, followed by fixation of the crossover byextension in a polymerase chain reaction. This process and many processvariants are described in several of the references above, e.g., inStemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751.

Similarly, nucleic acids can be recursively recombined in vivo, e.g., byallowing recombination to occur between nucleic acids in cells. Manysuch in vivo recombination formats are set forth in the references notedabove. Such formats optionally provide direct recombination betweennucleic acids of interest, or provide recombination between vectors,viruses, plasmids, etc., comprising the nucleic acids of interest, aswell as other formats. Details regarding such procedures are found inthe references noted above.

Whole genome recombination methods can also be used in which wholegenomes of cells or other organisms are recombined, optionally includingspiking of the genomic recombination mixtures with desired librarycomponents (e.g., genes corresponding to the pathways of the presentinvention). These methods have many applications, including those inwhich the identity of a target gene is not known. Details on suchmethods are found, e.g., in WO 98/31837 and in PCT/US99/15972. Thus, anyof these processes and techniques for recombination, recursiverecombination, and whole genome recombination, alone or in combination,can be used to generate the modified nucleic acid sequences and/ormodified gene fusion constructs of the present invention.

Synthetic recombination methods can also be used, in whicholigonucleotides corresponding to targets of interest are synthesizedand reassembled in PCR or ligation reactions which includeoligonucleotides which correspond to more than one parental nucleicacid, thereby generating new recombined nucleic acids. Oligonucleotidescan be made by standard nucleotide addition methods, or can be made,e.g., by tri-nucleotide synthetic approaches. Details regarding suchapproaches are found in the references noted above, including, e.g., WO00/42561, WO 01/23401, WO 00/42560, and, WO 00/42559.

In silico methods of recombination can be affected in which geneticalgorithms are used in a computer to recombine sequence strings whichcorrespond to homologous (or even non-homologous) nucleic acids. Theresulting recombined sequence strings are optionally converted intonucleic acids by synthesis of nucleic acids which correspond to therecombined sequences, e.g., in concert with oligonucleotidesynthesis/gene reassembly techniques. This approach can generate random,partially random or designed variants. Many details regarding in silicorecombination, including the use of genetic algorithms, geneticoperators and the like in computer systems, combined with generation ofcorresponding nucleic acids (and/or proteins), as well as combinationsof designed nucleic acids and/or proteins (e.g., based on cross-oversite selection) as well as designed, pseudo-random or randomrecombination methods are described in WO 00/42560 and WO 00/42559.

Many methods of accessing natural diversity, e.g., by hybridization ofdiverse nucleic acids or nucleic acid fragments to single-strandedtemplates, followed by polymerization and/or ligation to regeneratefull-length sequences, optionally followed by degradation of thetemplates and recovery of the resulting modified nucleic acids can besimilarly used. In one method employing a single-stranded template, thefragment population derived from the genomic library(ies) is annealedwith partial, or, often approximately full length ssDNA or RNAcorresponding to the opposite strand. Assembly of complex chimeric genesfrom this population is then mediated by nuclease-base removal ofnon-hybridizing fragment ends, polymerization to fill gaps between suchfragments and subsequent single stranded ligation. The parentalpolynucleotide strand can be removed by digestion (e.g., if RNA oruracil-containing), magnetic separation under denaturing conditions (iflabeled in a manner conducive to such separation) and other availableseparation/purification methods. Alternatively, the parental strand isoptionally co-purified with the chimeric strands and removed duringsubsequent screening and processing steps. Additional details regardingthis approach are found, e.g., in PCT/US01/06775.

In another approach, single-stranded molecules are converted todouble-stranded DNA (dsDNA) and the dsDNA molecules are bound to a solidsupport by ligand-mediated binding. After separation of unbound DNA, theselected DNA molecules are released from the support and introduced intoa suitable host cell to generate a library enriched sequences whichhybridize to the probe. A library produced in this manner provides adesirable substrate for further diversification using any of theprocedures described herein.

Any of the preceding general recombination formats can be practiced in areiterative fashion (e.g., one or more cycles of mutation/recombinationor other diversity generation methods, optionally followed by one ormore selection methods) to generate a more diverse set of recombinantnucleic acids.

Mutagenesis employing polynucleotide chain termination methods have alsobeen proposed (see e.g., U.S. Pat. No. 5,965,408 and the referencesabove), and can be applied to the present invention. In this approach,double stranded DNAs corresponding to one or more genes sharing regionsof sequence similarity are combined and denatured, in the presence orabsence of primers specific for the gene. The single strandedpolynucleotides are then annealed and incubated in the presence of apolymerase and a chain terminating reagent (e.g., ultraviolet, gamma orX-ray irradiation; ethidium bromide or other intercalators; DNA bindingproteins, such as single strand binding proteins, transcriptionactivating factors, or histones; polycyclic aromatic hydrocarbons;trivalent chromium or a trivalent chromium salt; or abbreviatedpolymerization mediated by rapid thermocycling; and the like), resultingin the production of partial duplex molecules. The partial duplexmolecules, e.g., containing partially extended chains, are thendenatured and reannealed in subsequent rounds of replication or partialreplication resulting in polynucleotides which share varying degrees ofsequence similarity and which are diversified with respect to thestarting population of DNA molecules. Optionally, the products, orpartial pools of the products, can be amplified at one or more stages inthe process. Polynucleotides produced by a chain termination method,such as described above, are suitable substrates for any other describedrecombination format.

Diversity also can be generated in nucleic acids or populations ofnucleic acids using a recombinational procedure termed “incrementaltruncation for the creation of hybrid enzymes” (“ITCHY”) described inOstermeier et al. (1999) Nature Biotech 17:1205. This approach can beused to generate an initial a library of variants which can optionallyserve as a substrate for one or more in vitro or in vivo recombinationmethods. See, also, Ostermeier et al. (1999) Proc. Natl. Acad. Sci. USA,96: 3562-67; Ostermeier et al. (1999), Biological and MedicinalChemistry 7: 2139-44.

Mutational methods which result in the alteration of individualnucleotides or groups of contiguous or non-contiguous nucleotides can befavorably employed to introduce nucleotide diversity into the nucleicacid sequences and/or gene fusion constructs of the present invention.Many mutagenesis methods are found in the above-cited references;additional details regarding mutagenesis methods can be found infollowing, which can also be applied to the present invention.

For example, error-prone PCR can be used to generate nucleic acidvariants. Using this technique, PCR is performed under conditions wherethe copying fidelity of the DNA polymerase is low, such that a high rateof point mutations is obtained along the entire length of the PCRproduct. Examples of such techniques are found in the references aboveand, e.g., in Leung et al. (1989) Technique 1:11-15 and Caldwell et al.(1992) PCR Methods Applic. 2:28-33. Similarly, assembly PCR can be used,in a process which involves the assembly of a PCR product from a mixtureof small DNA fragments. A large number of different PCR reactions canoccur in parallel in the same reaction mixture, with the products of onereaction priming the products of another reaction.

Oligonucleotide directed mutagenesis can be used to introducesite-specific mutations in a nucleic acid sequence of interest. Examplesof such techniques are found in the references above and, e.g., inReidhaar-Olson et al. (1988) Science 241:53-57. Similarly, cassettemutagenesis can be used in a process that replaces a small region of adouble stranded DNA molecule with a synthetic oligonucleotide cassettethat differs from the native sequence. The oligonucleotide can contain,e.g., completely and/or partially randomized native sequence(s).

Recursive ensemble mutagenesis is a process in which an algorithm forprotein mutagenesis is used to produce diverse populations ofphenotypically related mutants, members of which differ in amino acidsequence. This method uses a feedback mechanism to monitor successiverounds of combinatorial cassette mutagenesis. Examples of this approachare found in Arkin & Youvan (1992) Proc. Natl. Acad. Sci. USA89:7811-7815.

Exponential ensemble mutagenesis can be used for generatingcombinatorial libraries with a high percentage of unique and functionalmutants. Small groups of residues in a sequence of interest arerandomized in parallel to identify, at each altered position, aminoacids which lead to functional proteins. Examples of such procedures arefound in Delegrave & Youvan (1993) Biotechnology Research 11:1548-1552.

In vivo mutagenesis can be used to generate random mutations in anycloned DNA of interest by propagating the DNA, e.g., in a strain of E.coli that carries mutations in one or more of the DNA repair pathways.These “mutator” strains have a higher random mutation rate than that ofa wild-type parent. Propagating the DNA in one of these strains willeventually generate random mutations within the DNA. Such procedures aredescribed in the references noted above.

Other procedures for introducing diversity into a genome, e.g. abacterial, fungal, animal or plant genome can be used in conjunctionwith the above described and/or referenced methods. For example, inaddition to the methods above, techniques have been proposed whichproduce nucleic acid multimers suitable for transformation into avariety of species (see, e.g., U.S. Pat. No. 5,756,316 and thereferences above). Transformation of a suitable host with suchmultimers, consisting of genes that are divergent with respect to oneanother, (e.g., derived from natural diversity or through application ofsite directed mutagenesis, error prone PCR, passage through mutagenicbacterial strains, and the like), provides a source of nucleic aciddiversity for DNA diversification, e.g., by an in vivo recombinationprocess as indicated above.

Alternatively, a multiplicity of monomeric polynucleotides sharingregions of partial sequence similarity can be transformed into a hostspecies and recombined in vivo by the host cell. Subsequent rounds ofcell division can be used to generate libraries, members of which,include a single, homogenous population, or pool of monomericpolynucleotides. Alternatively, the monomeric nucleic acid can berecovered by standard techniques, e.g., PCR and/or cloning, andrecombined in any of the recombination formats, including recursiverecombination formats, described above.

Methods for generating multispecies expression libraries have beendescribed (in addition to the reference noted above, see, e.g., U.S.Pat. Nos. 5,783,431 and 5,824,485) and their use to identify proteinactivities of interest has been proposed (In addition to the referencesnoted above, see, U.S. Pat. No. 5,958,672. Multispecies expressionlibraries include, in general, libraries comprising cDNA or genomicsequences from a plurality of species or strains, operably linked toappropriate regulatory sequences, in an expression cassette. The cDNAand/or genomic sequences are optionally randomly ligated to furtherenhance diversity. The vector can be a shuttle vector suitable fortransformation and expression in more than one species of host organism,e.g., bacterial species, eukaryotic cells. In some cases, the library isbiased by preselecting sequences which encode a protein of interest, orwhich hybridize to a nucleic acid of interest. Any such libraries can beprovided as substrates for any of the methods herein described.

The above described procedures have been largely directed to increasingnucleic acid and/or encoded protein diversity. However, in many cases,not all of the diversity is useful, e.g., functional, and contributesmerely to increasing the background of variants that must be screened orselected to identify the few favorable variants. In some applications,it is desirable to preselect or prescreen libraries (e.g., an amplifiedlibrary, a genomic library, a cDNA library, a normalized library, etc.)or other substrate nucleic acids prior to diversification, e.g., byrecombination-based mutagenesis procedures, or to otherwise bias thesubstrates towards nucleic acids that encode functional products. Forexample, in the case of antibody engineering, it is possible to bias thediversity generating process toward antibodies with functional antigenbinding sites by taking advantage of in vivo recombination events priorto manipulation by any of the described methods. For example, recombinedCDRs derived from B cell cDNA libraries can be amplified and assembledinto framework regions (e.g., Jirholt et al. (1998) Gene 215: 471) priorto diversifying according to any of the methods described herein.

Libraries can be biased towards nucleic acids which encode proteins withdesirable enzyme activities. For example, after identifying a variantfrom a library which exhibits a specified activity, the variant can bemutagenized using any known method for introducing DNA alterations. Alibrary comprising the mutagenized homologues is then screened for adesired activity, which can be the same as or different from theinitially specified activity. An example of such a procedure is proposedin U.S. Pat. No. 5,939,250. Desired activities can be identified by anymethod known in the art. For example, WO 99/10539 proposes that genelibraries can be screened by combining extracts from the gene librarywith components obtained from metabolically rich cells and identifyingcombinations which exhibit the desired activity. It has also beenproposed (e.g., WO 98/58085) that clones with desired activities can beidentified by inserting bioactive substrates into samples of thelibrary, and detecting bioactive fluorescence corresponding to theproduct of a desired activity using a fluorescent analyzer, e.g., a flowcytometry device, a CCD, a fluorometer, or a spectrophotometer.

Libraries can also be biased towards nucleic acids which have specifiedcharacteristics, e.g., hybridization to a selected nucleic acid probe.For example, application WO 99/10539 proposes that polynucleotidesencoding a desired activity (e.g., an enzymatic activity, for example: alipase, an esterase, a protease, a glycosidase, a glycosyl transferase,a phosphatase, a kinase, an oxygenase, a peroxidase, a hydrolase, ahydratase, a nitrilase, a transaminase, an amidase or an acylase) can beidentified from among genomic DNA sequences in the following manner.Single stranded DNA molecules from a population of genomic DNA arehybridized to a ligand-conjugated probe. The genomic DNA can be derivedfrom either a cultivated or uncultivated microorganism, or from anenvironmental sample. Alternatively, the genomic DNA can be derived froma multicellular organism, or a tissue derived there from. Second strandsynthesis can be conducted directly from the hybridization probe used inthe capture, with or without prior release from the capture medium or bya wide variety of other strategies known in the art. Alternatively, theisolated single-stranded genomic DNA population can be fragmentedwithout further cloning and used directly in, e.g., arecombination-based approach, that employs a single-stranded template,as described above.

“Non-Stochastic” methods of generating nucleic acids and polypeptidesare found in WO 00/46344. These methods, including proposednon-stochastic polynucleotide reassembly and site-saturation mutagenesismethods be applied to the present invention as well. Random orsemi-random mutagenesis using doped or degenerate oligonucleotides isalso described in, e.g., Arkin and Youvan (1992) Biotechnology10:297-300; Reidhaar-Olson et al. (1991)Methods Enzymol. 208:564-86; Limand Sauer (1991) J. Mol. Biol. 219:359-76; Breyer and Sauer (1989) J.Biol. Chem. 264:13355-60); and U.S. Pat. Nos. 5,830,650 and 5,798,208,and EP Patent 0527809 B1.

It will readily be appreciated that any of the above describedtechniques suitable for enriching a library prior to diversification canalso be used to screen the products, or libraries of products, producedby the diversity generating methods. Any of the above described methodscan be practiced recursively or in combination to alter nucleic acids,e.g., meganuclease encoding polynucleotides.

The above references provide many mutational formats, includingrecombination, recursive recombination, recursive mutation andcombinations or recombination with other forms of mutagenesis, as wellas many modifications of these formats. Regardless of the diversitygeneration format that is used, the nucleic acids of the presentinvention can be recombined (with each other, or with related (or evenunrelated) sequences) to produce a diverse set of recombinant nucleicacids for use in the gene fusion constructs and modified gene fusionconstructs of the present invention, including, e.g., sets of homologousnucleic acids, as well as corresponding polypeptides.

Many of the above-described methodologies for generating modifiedpolynucleotides generate a large number of diverse variants of aparental sequence or sequences. In some embodiments, the modificationtechnique (e.g., some form of shuffling) is used to generate a libraryof variants that is then screened for a modified polynucleotide or poolof modified polynucleotides encoding some desired functional attribute,e.g., increased meganuclease activity.

For convenience and high throughput it will often be desirable toscreen/select for desired modified nucleic acids in a microorganism,e.g., a bacteria such as E. coli. On the other hand, screening in plantcells or plants can in some cases be preferable where the ultimate aimis to generate a modified nucleic acid for expression in a plant system.

In some preferred embodiments of the invention throughput is increasedby screening pools of host cells expressing different modified nucleicacids, either alone or as part of a gene fusion construct. Any poolsshowing significant activity can be deconvoluted to identify singlevariants expressing the desirable activity.

In high throughput assays, it is possible to screen up to severalthousand different variants in a single day. For example, each well of amicrotiter plate can be used to run a separate assay, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single variant.

In addition to fluidic approaches, it is possible, as mentioned above,simply to grow cells on media plates that select for the desiredenzymatic or metabolic function. This approach offers a simple andhigh-throughput screening method.

A number of well known robotic systems have also been developed forsolution phase chemistries useful in assay systems. These systemsinclude automated workstations like the automated synthesis apparatusdeveloped by Takeda Chemical Industries, LTD. (Osaka, Japan) and manyrobotic systems utilizing robotic arms (Zymate II, Zymark Corporation,Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.) which mimicthe manual synthetic operations performed by a scientist. Any of theabove devices are suitable for application to the present invention. Thenature and implementation of modifications to these devices (if any) sothat they can operate as discussed herein with reference to theintegrated system will be apparent to persons skilled in the relevantart.

High throughput screening systems are commercially available (see, e.g.,Zymark Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio;Beckman Instruments, Inc. Fullerton, Calif.; Precision Systems, Inc.,Natick, Mass., etc.). These systems typically automate entire proceduresincluding all sample and reagent pipetting, liquid dispensing, timedincubations, and final readings of the microplate in detector(s)appropriate for the assay. These configurable systems provide highthroughput and rapid start up as well as a high degree of flexibilityand customization.

The manufacturers of such systems provide detailed protocols for thevarious high throughput devices. Thus, for example, Zymark Corp.provides technical bulletins describing screening systems for detectingthe modulation of gene transcription, ligand binding, and the like.Microfluidic approaches to reagent manipulation have also beendeveloped, e.g., by Caliper Technologies (Mountain View, Calif.).

Yeast and Plants

Yeast, plants, plant cells, plant parts and seeds, and grain having themeganuclease sequences disclosed herein are provided. In specificembodiments, the yeast, plants and/or plant parts have stablyincorporated at least one heterologous meganuclease polypeptidedisclosed herein or an active variant or fragment thereof. Thus, yeast,plants, plant cells, plant parts and seed are provided which comprise atleast one heterologous meganuclease sequences of any one of SEQ ID NOs:14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 251,252, 253, 262, 272, 273, 274, 275, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 315, 316, 317, 318, 319, 320,330, 331, 332, 334, 335, 336, 337, 338, 339, 340, 341, 357, 358, 359,360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 370, 371, 390,391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402 or 403 or anyone of other variants disclosed herein, such as those in Example 3-23 ora biologically active fragment and/or variant of the meganucleasesequence. In specific embodiments, the meganuclease sequences arecharacterized as having meganuclease activity.

In specific embodiments, the heterologous polynucleotide in the plant orplant part is operably linked to a constitutive, tissue-preferred, orother promoter for expression in plants.

The yeast, plant cell, plant, plant part and seed can comprise any ofthe recognition sequence provided herein. For example, the recognitionsite can be selected from the group consisting of the LIG3-4 (SEQ ID NO:2), MHP77 (SEQ ID NO: 85), MS26 (SEQ ID NO: 269), MHP14 (SEQ ID NO:281), MP107 (SEQ ID NO: 328), ZM6.3 (SEQ ID NO: 355), ZM6.22V2 (SEQ IDNO: 388), TS21 (SEQ ID NO: 423) and/or TS14 (SEQ ID NO: 424) recognitionsequences or an active variant thereof.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which plants can be regenerated, plantcalli, plant clumps, and plant cells that are intact in plants or partsof plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like. Grain is intended to mean the mature seedproduced by commercial growers for purposes other than growing orreproducing the species. Progeny, variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced polynucleotides.

A transformed plant or transformed plant cell provided herein is one inwhich genetic alteration, such as transformation, has been affected asto a gene of interest, or is a plant or plant cell which is descendedfrom a plant or cell so altered and which comprises the alteration. A“transgene” is a gene that has been introduced into the genome by atransformation procedure. Accordingly, a “transgenic plant” is a plantthat contains a transgene, whether the transgene was introduced intothat particular plant by transformation or by breeding; thus,descendants of an originally-transformed plant are encompassed by thedefinition. A “control” or “control plant” or “control plant cell”provides a reference point for measuring changes in phenotype of thesubject plant or plant cell. A control plant or plant cell may comprise,for example: (a) a wild-type plant or cell, i.e., of the same genotypeas the starting material for the genetic alteration which resulted inthe subject plant or cell; (b) a plant or plant cell of the samegenotype as the starting material but which has been transformed with anull construct (i.e., with a construct which does not express thetransgene, such as a construct comprising a marker gene); (c) a plant orplant cell which is a non-transformed segregant among progeny of asubject plant or plant cell; (d) a plant or plant cell geneticallyidentical to the subject plant or plant cell but which is not exposed toconditions or stimuli that would induce expression of the transgene; or(e) the subject plant or plant cell itself, under conditions in whichthe construct is not expressed.

Plant cells that have been transformed to express a meganucleaseprovided herein can be grown into whole plants. The regeneration,development, and cultivation of plants from single plant protoplasttransformants or from various transformed explants is well known in theart. See, for example, McCormick et al. (1986) Plant Cell Reports5:81-84; Weissbach and Weissbach, In: Methods for Plant MolecularBiology, (Eds.), Academic Press, Inc. San Diego, Calif., (1988). Thisregeneration and growth process typically includes the steps ofselection of transformed cells, culturing those individualized cellsthrough the usual stages of embryonic development through the rootedplantlet stage. Transgenic embryos and seeds are similarly regenerated.The resulting transgenic rooted shoots are thereafter planted in anappropriate plant growth medium such as soil. Preferably, theregenerated plants are self-pollinated to provide homozygous transgenicplants. Otherwise, pollen obtained from the regenerated plants iscrossed to seed-grown plants of agronomically important lines.Conversely, pollen from plants of these important lines is used topollinate regenerated plants. Two or more generations may be grown toensure that expression of the desired phenotypic characteristic isstably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the compositions presented herein provide transformedseed (also referred to as “transgenic seed”) having a polynucleotideprovided herein, for example, a target site, stably incorporated intotheir genome.

The meganuclease sequences and active variant and fragments thereofdisclosed herein may be used for transformation of any plant species,including, but not limited to, monocots and dicots. Examples of plantspecies of interest include, but are not limited to, corn (Zea mays),Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly thoseBrassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis), and Poplar and Eucalyptus. In specificembodiments, plants of the present invention are crop plants (forexample, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower,peanut, sorghum, wheat, millet, tobacco, etc.). In other embodiments,corn and soybean plants are optimal, and in yet other embodiments cornplants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

Non-limiting examples of compositions and methods disclosed herein areas follows:

-   1. An isolated or recombinant polynucleotide comprising a nucleotide    sequence encoding a meganuclease polypeptide, said polypeptide    comprising:    -   a) an amino acid sequence having at least one amino acid        modification at an amino acid position corresponding to a        position of SEQ ID NO: 1 selected from the group consisting of        positions 2, 12, 16, 22, 23, 31, 36, 43, 50, 56, 58, 59, 62, 71,        72, 73, 80, 81, 82, 86, 91, 95, 98, 103, 113, 114, 116, 117,        118, 121, 124, 128, 129, 131, 147, 151, 153, 159, 160, 161, 162,        163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,        176, 177, 178, 179, 180, 182, 183, 184, 185, 186, 187, 188, 189,        190, 191, 192, 194, 195, 196, 197, 200, 203, 204, 209, 222, 232,        236, 237, 246, 254, 258, 267, 278, 281, 282, 289, 308, 311, 312,        316, 318, 319, 334, 339, 340, 342, 345, 346, 348 and        combinations thereof, or,    -   b) an amino acid sequence having at least 1, 2, 3, 4, 5, 6, 7,        8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,        40, 41, 42, 43 or 44 of any of the amino acid modification of        (a);-   2. The isolated or recombinant polynucleotide of embodiment 1,    wherein said nucleotide sequence encodes a meganuclease polypeptide    having at least 80%, 81, %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,    90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity    to SEQ ID NO: 1.-   3. The isolated or recombinant polynucleotide of embodiment 1,    wherein said at least one amino acid modification comprises;    -   a) an aspartic acid (D) at a position corresponding to amino        acid position 2 in SEQ ID NO: 1;    -   b) a histidine (H) at a position corresponding to amino acid        position 12 in SEQ ID NO: 1;    -   c) an isoleucine (I) at a position corresponding to amino acid        position 16 in SEQ ID NO: 1;    -   d) a cysteine (C) at a position corresponding to amino acid        position 22 in SEQ ID NO: 1;    -   e) a leucine (L) at a position corresponding to amino acid        position 23 in SEQ ID NO: 1;    -   f) an arginine (R) at a position corresponding to amino acid        position 31 in SEQ ID NO: 1;    -   g) an asparagine (N) at a position corresponding to amino acid        position 36 in SEQ ID NO: 1;    -   h) a leucine (L) at a position corresponding to amino acid        position 43 in SEQ ID NO: 1;    -   i) an arginine (R) or lysine (K) at a position corresponding to        amino acid position 50 in SEQ ID NO: 1;    -   j) a leucine (L) at a position corresponding to amino acid        position 56 in SEQ ID NO: 1;    -   k) an isoleucine (I) at a position corresponding to amino acid        position 58 in SEQ ID NO: 1;    -   l) a histidine (H) or alanine (A) at a position corresponding to        amino acid position 59 in SEQ ID NO: 1;    -   m) a valine (V) at a position corresponding to amino acid        position 62 in SEQ ID NO: 1;    -   n) a lysine (K) at a position corresponding to amino acid        position 71 in SEQ ID NO: 1;    -   o) a threonine (T) at a position corresponding to amino acid        position 72 in SEQ ID NO: 1;    -   p) an alanine (A) at a position corresponding to amino acid        position 73 in SEQ ID NO: 1;    -   q) an arginine (R) at a position corresponding to amino acid        position 80 in SEQ ID NO: 1;    -   r) a lysine (K) at a position corresponding to amino acid        position 81 in SEQ ID NO: 1;    -   s) an arginine (R) at a position corresponding to amino acid        position 82 in SEQ ID NO: 1;    -   t) an aspartic acid (D) at a position corresponding to amino        acid position 86 in SEQ ID NO: 1;    -   u) an isoleucine (I) at a position corresponding to amino acid        position 91 in SEQ ID NO: 1;    -   v) an isoleucine (I) at a position corresponding to amino acid        position 95 in SEQ ID NO: 1;    -   w) an arginine (R) at a position corresponding to amino acid        position 98 in SEQ ID NO: 1;    -   x) a valine (V) at a position corresponding to amino acid        position 103 in SEQ ID NO: 1;    -   y) a serine (S) at a position corresponding to amino acid        position 113 in SEQ ID NO: 1;    -   z) a proline (P) at a position corresponding to amino acid        position 114 in SEQ ID NO: 1;    -   aa) an arginine (R) at a position corresponding to amino acid        position 116 in SEQ ID NO: 1;    -   bb) a glycine (G) at a position corresponding to amino acid        position 117 in SEQ ID NO: 1;    -   cc) a threonine (T) at a position corresponding to amino acid        position 118 in SEQ ID NO: 1;    -   dd) a an glycine (G) at a position corresponding to amino acid        position 121 in SEQ ID NO: 1;    -   ee) an arginine (R) at a position corresponding to amino acid        position 124 in SEQ ID NO: 1;    -   ff) a cysteine (C) at a position corresponding to amino acid        position 128 in SEQ ID NO: 1;    -   gg) an alanine (A) at a position corresponding to amino acid        position 129 in SEQ ID NO: 1;    -   hh) an arginine (R) at a position corresponding to amino acid        position 131 in SEQ ID NO: 1;    -   ii) a serine (S) at a position corresponding to amino acid        position 147 in SEQ ID NO: 1;    -   jj) an alanine (A) at a position corresponding to amino acid        position 151 in SEQ ID NO: 1;    -   kk) a leucine (L) or a methionine (M) at a position        corresponding to amino acid position 153 in SEQ ID NO: 1;    -   ll) a tryptophan (W) at a position corresponding to amino acid        position 159 in SEQ ID NO: 1;    -   mm) a glutamic acid (E) at a position corresponding to amino        acid position 160 in SEQ ID NO: 1;    -   nn) a valine (V) at a position corresponding to amino acid        position 161 in SEQ ID NO: 1;    -   oo) a tyrosine (Y) at a position corresponding to amino acid        position 162 in SEQ ID NO: 1;    -   pp) an arginine (R) at a position corresponding to amino acid        position 163 in SEQ ID NO: 1;    -   qq) a histidine (H) at a position corresponding to amino acid        position 164 in SEQ ID NO: 1;    -   rr) a leucine (L) at a position corresponding to amino acid        position 165 in SEQ ID NO: 1;    -   ss) an arginine (R) at a position corresponding to amino acid        position 166 in SEQ ID NO: 1;    -   tt) a histidine (H) at a position corresponding to amino acid        position 167 in SEQ ID NO: 1;    -   uu) a proline (P) at a position corresponding to amino acid        position 168 in SEQ ID NO: 1;    -   vv) an alanine (A) at a position corresponding to amino acid        position 169 in SEQ ID NO: 1;    -   ww) a proline (P) at a position corresponding to amino acid        position 170 in SEQ ID NO: 1;    -   xx) a histidine (H) at a position corresponding to amino acid        position 171 in SEQ ID NO: 1;    -   yy) a proline (P) at a position corresponding to amino acid        position 172 in SEQ ID NO: 1;    -   zz) an arginine (R) at a position corresponding to amino acid        position 173 in SEQ ID NO: 1;    -   aaa) a leucine (L) at a position corresponding to amino acid        position 174 in SEQ ID NO: 1;    -   bbb) a proline (P) at a position corresponding to amino acid        position 175 in SEQ ID NO: 1;    -   ccc) a glutamine (Q) at a position corresponding to amino acid        position 176 in SEQ ID NO: 1;    -   ddd) an alanine (A) at a position corresponding to amino acid        position 177 in SEQ ID NO: 1;    -   eee) an arginine (R) at a position corresponding to amino acid        position 178 in SEQ ID NO: 1;    -   fff) a valine (V) at a position corresponding to amino acid        position 179 in SEQ ID NO: 1;    -   ggg) a glutamine (Q) at a position corresponding to amino acid        position 180 in SEQ ID NO: 1;    -   hhh) a valine (V) at a position corresponding to amino acid        position 182 in SEQ ID NO: 1;    -   iii) a proline (P) at a position corresponding to amino acid        position 183 in SEQ ID NO: 1;    -   jjj) a lysine (K) at a position corresponding to amino acid        position 184 in SEQ ID NO: 1;    -   kkk) a threonine (T) or a histidine (H) at a position        corresponding to amino acid position 185 in SEQ ID NO: 1;    -   lll) a serine (S) at a position corresponding to amino acid        position 186 in SEQ ID NO: 1;    -   mmm) a glutamic acid (E) at a position corresponding to amino        acid position 187 in SEQ ID NO: 1;    -   nnn) a leucine (L) at a position corresponding to amino acid        position 188 in SEQ ID NO: 1;    -   ooo) a glutamic acid (E) at a position corresponding to amino        acid position 189 in SEQ ID NO: 1;    -   ppp) a glutamine (Q) at a position corresponding to amino acid        position 190 in SEQ ID NO: 1;    -   qqq) a leucine (L) at a position corresponding to amino acid        position 191 in SEQ ID NO: 1;    -   rrr) a proline (P) at a position corresponding to amino acid        position 194 in SEQ ID NO: 1;    -   sss) a lysine (K) at a position corresponding to amino acid        position 195 in SEQ ID NO: 1;    -   ttt) a serine (S) at a position corresponding to amino acid        position 196 in SEQ ID NO: 1;    -   uuu) a phenylalanine (F) at a position corresponding to amino        acid position 197 in SEQ ID NO: 1;    -   vvv) an isoleucine (I) at a position corresponding to amino acid        position 200 in SEQ ID NO: 1;    -   www) a valine (V) at a position corresponding to amino acid        position 203 in SEQ ID NO: 1;    -   xxx) a leucine (L) at a position corresponding to amino acid        position 204 in SEQ ID NO: 1;    -   yyy) a cysteine (C) at a position corresponding to amino acid        position 209 in SEQ ID NO: 1;    -   zzz) a leucine (L) at a position corresponding to amino acid        position 222 in SEQ ID NO: 1;    -   aaaa) an isoleucine (I) at a position corresponding to amino        acid position 232 in SEQ ID NO: 1;    -   bbbb) a serine (S) at a position corresponding to amino acid        position 236 in SEQ ID NO: 1;    -   cccc) a leucine (L) or an arginine (R) at a position        corresponding to amino acid position 237 in SEQ ID NO: 1;    -   dddd) a histidine (H) at a position corresponding to amino acid        position 246 in SEQ ID NO: 1;    -   eeee) an isoleucine (I) at a position corresponding to amino        acid position 254 in SEQ ID NO: 1;    -   ffff) a serine (S) at a position corresponding to amino acid        position 258 in SEQ ID NO: 1;    -   gggg) an arginine (R) at a position corresponding to amino acid        position 267 in SEQ ID NO: 1;    -   hhhh) an isoleucine (I) at a position corresponding to amino        acid position 278 in SEQ ID NO: 1;    -   iiii) a tyrosine (Y) at a position corresponding to amino acid        position 281 in SEQ ID NO: 1;    -   jjjj) a phenylalanine (F) at a position corresponding to amino        acid position 282 in SEQ ID NO: 1;    -   kkkk) a threonine (T) at a position corresponding to amino acid        position 289 in SEQ ID NO: 1;    -   llll) a glycine (G) at a position corresponding to amino acid        position 308 in SEQ ID NO: 1;    -   mmmm) an arginine (R) at a position corresponding to amino acid        position 311 in SEQ ID NO: 1;    -   nnnn) an alanine (A) at a position corresponding to amino acid        position 312 in SEQ ID NO: 1;    -   oooo) an alanine (A) at a position corresponding to amino acid        position 316 in SEQ ID NO: 1;    -   pppp) an arginine (R) at a position corresponding to amino acid        position 318 in SEQ ID NO: 1    -   qqqq) an alanine (A) at a position corresponding to amino acid        position 334 in SEQ ID NO: 1;    -   rrrr) a phenylalanine (F) at a position corresponding to amino        acid position 339 in SEQ ID NO: 1;    -   ssss) a glycine (G) or a leucine (L) at a position corresponding        to amino acid position 340 in SEQ ID NO: 1;    -   tttt) a serine (S) at a position corresponding to amino acid        position 342 in SEQ ID NO: 1;    -   uuuu) an asparagine (N) at a position corresponding to amino        acid position 345 in SEQ ID NO: 1;    -   vvvv) an asparagine (N) at a position corresponding to amino        acid position 346 in SEQ ID NO: 1;    -   wwww) an asparagine (N) at a position corresponding to amino        acid position 348 in SEQ ID NO: 1; or,    -   xxxx) any combination of a) to wwww).-   4. The isolated or recombinant polynucleotide of embodiment 1,    wherein said nucleotide sequence encodes a meganuclease polypeptide,    wherein said polypeptide further comprises:    -   a) an aspartic acid (D) at a position corresponding to amino        acid position 2 in SEQ ID NO: 1;    -   b) a histidine (H) at a position corresponding to amino acid        position 12 in SEQ ID NO: 1;    -   c) an isoleucine (I) at a position corresponding to amino acid        position 16 in SEQ ID NO: 1;    -   d) a serine (S) or an alanine (A) at a position corresponding to        amino acid position 19 in SEQ ID NO: 1;    -   e) a cysteine (C) at a position corresponding to amino acid        position 22 in SEQ ID NO: 1;    -   f) a leucine (L) at a position corresponding to amino acid        position 23 in SEQ ID NO: 1;    -   g) a methionine (M) at a position corresponding to amino acid        position 24 in SEQ ID NO: 1;    -   h) an arginine (R) or an alanine (A) at a position corresponding        to amino acid position 28 in SEQ ID NO: 1;    -   i) an arginine (R), alanine (A), glutamine (Q), cysteine (C),        glycine (G), serine (S), threonine (T), leucine (L), glutamic        acid (E), or a proline (P) at a position corresponding to amino        acid position 30 in SEQ ID NO: 1;    -   j) an arginine (R) at a position corresponding to amino acid        position 31 in SEQ ID NO: 1;    -   k) an arginine (R), alanine (A), lysine (K) glutamine (Q),        glycine (G) or a leucine (L) at a position corresponding to        amino acid position 32 in SEQ ID NO: 1;    -   l) an asparagine (N) at a position corresponding to amino acid        position 36 in SEQ ID NO: 1;    -   m) a leucine (L) at a position corresponding to amino acid        position 43 in SEQ ID NO: 1;    -   n) an arginine (R) or lysine (K) at a position corresponding to        amino acid position 50 in SEQ ID NO: 1;    -   o) an isoleucine (I) or a leucine (L) at a position        corresponding to amino acid position 54 in SEQ ID NO: 1;    -   p) a leucine (L) at a position corresponding to amino acid        position 56 in SEQ ID NO: 1;    -   q) a glutamic acid (E) at a position corresponding to amino acid        position 57 in SEQ ID NO: 1;    -   r) an isoleucine (I) at a position corresponding to amino acid        position 58 in SEQ ID NO: 1;    -   s) a histidine (H) or alanine (A) at a position corresponding to        amino acid position 59 in SEQ ID NO: 1;    -   t) a valine (V) at a position corresponding to amino acid        position 62 in SEQ ID NO: 1;    -   u) a lysine (K) at a position corresponding to amino acid        position 71 in SEQ ID NO: 1;    -   v) a threonine (T) at a position corresponding to amino acid        position 72 in SEQ ID NO: 1;    -   w) an alanine (A) at a position corresponding to amino acid        position 73 in SEQ ID NO: 1;    -   x) a glycine (G) at a position corresponding to amino acid        position 79 in SEQ ID NO: 1;    -   y) an arginine (R) at a position corresponding to amino acid        position 80 in SEQ ID NO: 1;    -   z) a lysine (K) at a position corresponding to amino acid        position 81 in SEQ ID NO: 1;    -   aa) an arginine (R) at a position corresponding to amino acid        position 82 in SEQ ID NO: 1;    -   bb) an aspartic acid (D) at a position corresponding to amino        acid position 86 in SEQ ID NO: 1;    -   cc) a leucine (L) at a position corresponding to amino acid        position 87 in SEQ ID NO: 1;    -   dd) an isoleucine (I) at a position corresponding to amino acid        position 91 in SEQ ID NO: 1;    -   ee) an isoleucine (I) at a position corresponding to amino acid        position 95 in SEQ ID NO: 1;    -   ff) an arginine (R) at a position corresponding to amino acid        position 98 in SEQ ID NO: 1;    -   gg) a valine (V) at a position corresponding to amino acid        position 103 in SEQ ID NO: 1;    -   hh) an alanine (A) at a position corresponding to amino acid        position 105 in SEQ ID NO: 1;    -   ii) an arginine (R) at a position corresponding to amino acid        position 111 in SEQ ID NO: 1;    -   jj) a serine (S) at a position corresponding to amino acid        position 113 in SEQ ID NO: 1;    -   kk) a proline (P) at a position corresponding to amino acid        position 114 in SEQ ID NO: 1;    -   ll) an arginine (R) at a position corresponding to amino acid        position 116 in SEQ ID NO: 1;    -   mm) a an glycine (G) at a position corresponding to amino acid        position 117 in SEQ ID NO: 1;    -   nn) a threonine (T) at a position corresponding to amino acid        position 118 in SEQ ID NO: 1;    -   oo) a an glycine (G) at a position corresponding to amino acid        position 121 in SEQ ID NO: 1;    -   pp) an arginine (R) at a position corresponding to amino acid        position 124 in SEQ ID NO: 1;    -   qq) a cysteine (C) at a position corresponding to amino acid        position 128 in SEQ ID NO: 1;    -   rr) an alanine (A) at a position corresponding to amino acid        position 129 in SEQ ID NO: 1;    -   ss) an arginine (R) at a position corresponding to amino acid        position 131 in SEQ ID NO: 1;    -   tt) a valine (V) at a position corresponding to amino acid        position 132 in SEQ ID NO: 1;    -   uu) a serine (S) at a position corresponding to amino acid        position 147 in SEQ ID NO: 1;    -   vv) an alanine (A) at a position corresponding to amino acid        position 151 in SEQ ID NO: 1;    -   ww) a leucine (L) or a methionine (M) at a position        corresponding to amino acid position 153 in SEQ ID NO: 1;    -   xx) a tryptophan (W) at a position corresponding to amino acid        position 159 in SEQ ID NO: 1;    -   yy) a glutamic acid (E) at a position corresponding to amino        acid position 160 in SEQ ID NO: 1;    -   zz) a valine (V) at a position corresponding to amino acid        position 161 in SEQ ID NO: 1;    -   aaa) a tyrosine (Y) at a position corresponding to amino acid        position 162 in SEQ ID NO: 1;    -   bbb) an arginine (R) at a position corresponding to amino acid        position 163 in SEQ ID NO: 1;    -   ccc) a histidine (H) at a position corresponding to amino acid        position 164 in SEQ ID NO: 1;    -   ddd) a leucine (L) at a position corresponding to amino acid        position 165 in SEQ ID NO: 1;    -   eee) an arginine (R) at a position corresponding to amino acid        position 166 in SEQ ID NO: 1;    -   fff) a histidine (H) at a position corresponding to amino acid        position 167 in SEQ ID NO: 1;    -   ggg) a proline (P) at a position corresponding to amino acid        position 168 in SEQ ID NO: 1;    -   hhh) an alanine (A) at a position corresponding to amino acid        position 169 in SEQ ID NO: 1;    -   iii) a proline (P) at a position corresponding to amino acid        position 170 in SEQ ID NO: 1;    -   jjj) a histidine (H) at a position corresponding to amino acid        position 171 in SEQ ID NO: 1;    -   kkk) a proline (P) at a position corresponding to amino acid        position 172 in SEQ ID NO: 1;    -   lll) an arginine (R) at a position corresponding to amino acid        position 173 in SEQ ID NO: 1;    -   mmm) a leucine (L) at a position corresponding to amino acid        position 174 in SEQ ID NO: 1;    -   nnn) a proline (P) at a position corresponding to amino acid        position 175 in SEQ ID NO: 1;    -   ooo) a glutamine (Q) at a position corresponding to amino acid        position 176 in SEQ ID NO: 1;    -   ppp) an alanine (A) at a position corresponding to amino acid        position 177 in SEQ ID NO: 1;    -   qqq) an arginine (R) at a position corresponding to amino acid        position 178 in SEQ ID NO: 1;    -   rrr) a valine (V) at a position corresponding to amino acid        position 179 in SEQ ID NO: 1;    -   sss) a glutamine (Q) at a position corresponding to amino acid        position 180 in SEQ ID NO: 1;    -   ttt) a valine (V) at a position corresponding to amino acid        position 182 in SEQ ID NO: 1;    -   uuu) a proline (P) at a position corresponding to amino acid        position 183 in SEQ ID NO: 1;    -   vvv) a lysine (K) at a position corresponding to amino acid        position 184 in SEQ ID NO: 1;    -   www) a threonine (T) or a histidine (H) at a position        corresponding to amino acid position 185 in SEQ ID NO: 1;    -   xxx) a serine (S) at a position corresponding to amino acid        position 186 in SEQ ID NO: 1;    -   yyy) a glutamic acid (E) at a position corresponding to amino        acid position 187 in SEQ ID NO: 1;    -   zzz) a leucine (L) at a position corresponding to amino acid        position 188 in SEQ ID NO: 1;    -   aaaa) a glutamic acid (E) at a position corresponding to amino        acid position 189 in SEQ ID NO: 1;    -   bbbb) a glutamine (Q) at a position corresponding to amino acid        position 190 in SEQ ID NO: 1;    -   cccc) a leucine (L) at a position corresponding to amino acid        position 191 in SEQ ID NO: 1;    -   dddd) an amino acid deletion at a position corresponding to        amino acid position 192 in SEQ ID NO: 1;    -   eeee) a proline (P) at a position corresponding to amino acid        position 194 in SEQ ID NO: 1;    -   ffff) a lysine (K) at a position corresponding to amino acid        position 195 in SEQ ID NO: 1;    -   gggg) a serine (S) at a position corresponding to amino acid        position 196 in SEQ ID NO: 1;    -   hhhh) a phenylalanine (F) at a position corresponding to amino        acid position 197 in SEQ ID NO: 1;    -   iiii) an isoleucine (I) at a position corresponding to amino        acid position 200 in SEQ ID NO: 1;    -   jjjj) a valine (V) at a position corresponding to amino acid        position 203 in SEQ ID NO: 1;    -   kkkk) a leucine (L) at a position corresponding to amino acid        position 204 in SEQ ID NO: 1;    -   llll) an alanine (A) or a serine (S) at a position corresponding        to amino acid position 206 in SEQ ID NO: 1;    -   mmmm) a cysteine (C) at a position corresponding to amino acid        position 209 in SEQ ID NO: 1;    -   nnnn) a leucine (L) at a position corresponding to amino acid        position 222 in SEQ ID NO: 1;    -   oooo) a methionine (M) at a position corresponding to amino acid        position 211 in SEQ ID NO: 1;    -   pppp) an isoleucine (I) at a position corresponding to amino        acid position 232 in SEQ ID NO: 1;    -   qqqq) a serine (S) at a position corresponding to amino acid        position 236 in SEQ ID NO: 1;    -   rrrr) a leucine (L) or an arginine (R) at a position        corresponding to amino acid position 237 in SEQ ID NO: 1;    -   ssss) an isoleucine (I) or a leucine (L) at a position        corresponding to amino acid position 241 in SEQ ID NO: 1;    -   tttt) a glutamic acid (E) at a position corresponding to amino        acid position 244 in SEQ ID NO: 1;    -   uuuu) a histidine (H) at a position corresponding to amino acid        position 246 in SEQ ID NO: 1;    -   vvvv) an aspartic acid (D) or histidine (H) at a position        corresponding to amino acid position 253 in SEQ ID NO: 1;    -   wwww) an isoleucine (I) at a position corresponding to amino        acid position 254 in SEQ ID NO: 1;    -   xxxx) a serine (S) at a position corresponding to amino acid        position 258 in SEQ ID NO: 1;    -   yyyy) an arginine (R) at a position corresponding to amino acid        position 267 in SEQ ID NO: 1;    -   zzzz) an isoleucine (I) at a position corresponding to amino        acid position 278 in SEQ ID NO: 1;    -   aaaaa) a tyrosine (Y) at a position corresponding to amino acid        position 281 in SEQ ID NO: 1;    -   bbbbb) a phenylalanine (F) at a position corresponding to amino        acid position 282 in SEQ ID NO: 1;    -   ccccc) a threonine (T) at a position corresponding to amino acid        position 289 in SEQ ID NO: 1;    -   ddddd) an alanine (A) at a position corresponding to amino acid        position 292 in SEQ ID NO: 1;    -   eeeee) a glycine (G) at a position corresponding to amino acid        position 308 in SEQ ID NO: 1;    -   fffff) an arginine (R) at a position corresponding to amino acid        position 311 in SEQ ID NO: 1;    -   ggggg) an alanine (A) at a position corresponding to amino acid        position 312 in SEQ ID NO: 1;    -   hhhhh) an alanine (A) at a position corresponding to amino acid        position 316 in SEQ ID NO: 1;    -   iiiii) an arginine (R) at a position corresponding to amino acid        position 318 in SEQ ID NO: 1    -   jjjjj) a valine (V) at a position corresponding to amino acid        position 319 in SEQ ID NO: 1;    -   kkkkk) an alanine (A) at a position corresponding to amino acid        position 334 in SEQ ID NO: 1;    -   lllll) a phenylalanine (F) at a position corresponding to amino        acid position 339 in SEQ ID NO: 1;    -   mmmmm) a glycine (G) or a leucine (L) at a position        corresponding to amino acid position 340 in SEQ ID NO: 1;    -   nnnnn) a serine (S) at a position corresponding to amino acid        position 342 in SEQ ID NO: 1;    -   ooooo) an asparagine (N) at a position corresponding to amino        acid position 345 in SEQ ID NO: 1;    -   ppppp) an asparagine (N) at a position corresponding to amino        acid position 346 in SEQ ID NO: 1; or,    -   qqqqq) an asparagine (N) at a position corresponding to amino        acid position 348 in SEQ ID NO: 1; or,    -   rrrrr) any combination of a) to qqqqq).-   5. The isolated or recombinant polynucleotide of embodiment 1,    wherein said nucleotide sequence encodes a meganuclease polypeptide    selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17,    18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,    35, 36, 37, 38, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,    100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,    113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,    126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,    139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,    152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,    165, 166, 167, 251, 252, 253, 272, 273, 274, 275, 272, 273, 274,    275, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295,    296, 297, 298, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339,    340, 341, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367,    368, 369, 370, 371, 390, 391, 392, 393, 394, 395, 396, 397, 398,    399, 400, 401, 402, 403, 430, 431, 432 and 433.-   6. The isolated or recombinant polynucleotide of embodiment 1,    wherein said nucleotide sequence encodes a meganuclease polypeptide,    wherein the polypeptide is capable of recognizing and cleaving a    meganuclease recognition sequence selected from the group consisting    of SEQ ID NO: 2 (LIG3-4), SEQ ID NO: 85 (MHP77), SEQ ID NO: 269    (MS26), SEQ ID NO: 281 (MHP14), SEQ ID NO: 331(MP107), SEQ ID NO:    358 (ZM6.3), SEQ ID NO: 390 (ZM6.22v2), SEQ ID NO: 423 or SEQ ID NO:    424.-   7. The isolated or recombinant polynucleotide of embodiment 1,    wherein said nucleotide sequence encodes a meganuclease polypeptide,    wherein said polypeptide has an increased meganuclease activity when    compared to a control meganuclease that lacks said amino acid    modification.-   8. The isolated or recombinant polynucleotide of embodiment 7,    wherein said control meganuclease is selected from the group of SEQ    ID NO: 1 (LIG3-4), SEQ ID NO: 86 (MHP77), SEQ ID NO: 250 (MHP77.3),    SEQ ID NO: 270 (MS26+), SEQ ID NO: 271, SEQ ID NO: 282 (MHP14), SEQ    ID NO: 283 (MHP14+), SEQ ID NO: 329 (MP107), SEQ ID NO: 356 (ZM6.3),    SEQ ID NO: 389 (ZM6.22v2), SEQ ID NO: 429 or SEQ ID NO: 435.-   9. The isolated or recombinant polynucleotide of embodiment 7,    wherein the increased meganuclease activity is evidenced by:    -   a) a higher yeast assay score when compared to the control        meganuclease that lacks said amino acid modification; or,    -   b) a higher target site mutation rate when compared to the        control meganuclease that lacks said amino acid modification;        or,    -   c) a higher in-vitro cutting when compared to the control        meganuclease that lacks said amino acid modification; or,    -   d) any combination of (a), (b) and (c).-   10. The isolated or recombinant polynucleotide of embodiment 1,    further comprising a nucleotide sequence encoding a N-terminal    nuclear transit peptide.-   11. The isolated or recombinant polynucleotide of embodiment 1,    further comprising a nucleotide sequence encoding a C-terminal    histidine tag.-   12. The isolated or recombinant polynucleotide of embodiment 7,    wherein the increased meganuclease activity is determined at 16° C.,    24° C., 28° C., 30° C. or 37° C.-   13. A recombinant DNA construct, comprising the isolated or    recombinant polynucleotide of embodiment 1.-   14. The recombinant DNA construct of embodiment 13, further    comprising a promoter operably linked to said polynucleotide.-   15. The recombinant DNA construct of embodiment 14, wherein said    promoter is heterologous with respect to said polynucleotide or said    promoter is homologous with respect to said polynucleotide.-   16. A cell comprising at least one polynucleotide of embodiment 1 or    the recombinant DNA construct of embodiment 13, wherein said    polynucleotide is heterologous to the cell.-   17. The cell of embodiment 16, wherein said cell is a yeast cell.-   18. The cell of embodiment 16, wherein said cell is a plant cell.-   19. The cell of embodiment 16, wherein said polynucleotide or said    recombinant DNA construct is stably incorporated into the genome of    said plant cell.-   20. The cell of embodiment 16, wherein said polynucleotide or said    recombinant DNA construct is stably incorporated into the    chloroplast genome of said plant cell.-   21. The cell of embodiment 18, wherein said plant cell is from a    monocot.-   22. The cell of embodiment 21 wherein said monocot is maize, wheat,    rice, barley, sugarcane, sorghum, or rye.-   23. The cell of embodiment 18, wherein said plant cell is from a    dicot.-   24. The cell of embodiment 23, wherein the dicot is soybean,    Brassica, sunflower, cotton, or alfalfa.-   25. A plant comprising a plant cell of embodiment 18.-   26. A plant explant comprising a plant cell of embodiment 18.-   27. The plant, the explant or the plant cell of embodiment 26,    wherein said plant, explant or plant cell exhibits an increased    meganuclease activity when compared to a plant, explant or plant    cell of the same species, strain or cultivar that does not comprise    at least one polynucleotide of embodiments 1.-   28. A transgenic seed produced by the plant of embodiment 25.-   29. An isolated polypeptide having meganuclease activity, said    polypeptide comprising:    -   a) an amino acid sequence having at least one amino acid        modification at an amino acid position corresponding to a        position of SEQ ID NO: 1 selected from the group consisting of        positions 2, 12, 16, 22, 23, 31, 36, 43, 50, 56, 58, 59, 62, 71,        72, 73, 80, 81, 82, 86, 91, 95, 98, 103, 113, 114, 116, 117,        118, 121, 124, 128, 129, 131, 147, 151, 153, 159, 160, 161, 162,        163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,        176, 177, 178, 179, 180, 182, 183, 184, 185, 186, 187, 188, 189,        190, 191, 192, 194, 195, 196, 197, 200, 203, 204, 209, 222, 232,        236, 237, 246, 254, 258, 267, 278, 281, 282, 289, 308, 311, 312,        316, 318, 319, 334, 339, 340, 342, 345, 346, 348 and        combinations thereof; or,    -   b) an amino acid sequence having at least 1, 2, 3, 4, 5, 6, 7,        8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,        24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,        40, 41, 42, 43 or 44 of any of the amino acid modification of        (a);-   30. The isolated polypeptide of embodiment 29, wherein said    polypeptide has at least 80% sequence identity to SEQ ID NO: 1.-   31. The isolated polypeptide of embodiment 29, wherein said at least    one amino acid modification comprises:    -   a) an aspartic acid (D) at a position corresponding to amino        acid position 2 in SEQ ID NO: 1;    -   b) a histidine (H) at a position corresponding to amino acid        position 12 in SEQ ID NO: 1;    -   c) an isoleucine (I) at a position corresponding to amino acid        position 16 in SEQ ID NO: 1;    -   d) a cysteine (C) at a position corresponding to amino acid        position 22 in SEQ ID NO: 1;    -   e) a leucine (L) at a position corresponding to amino acid        position 23 in SEQ ID NO: 1;    -   f) an arginine (R) at a position corresponding to amino acid        position 31 in SEQ ID NO: 1;    -   g) an asparagine (N) at a position corresponding to amino acid        position 36 in SEQ ID NO: 1;    -   h) a leucine (L) at a position corresponding to amino acid        position 43 in SEQ ID NO: 1;    -   i) an arginine (R) or lysine (K) at a position corresponding to        amino acid position 50 in SEQ ID NO: 1;    -   j) a leucine (L) at a position corresponding to amino acid        position 56 in SEQ ID NO: 1;    -   k) an isoleucine (I) at a position corresponding to amino acid        position 58 in SEQ ID NO: 1;    -   l) a histidine (H) or alanine (A) at a position corresponding to        amino acid position 59 in SEQ ID NO: 1;    -   m) a valine (V) at a position corresponding to amino acid        position 62 in SEQ ID NO: 1;    -   n) a lysine (K) at a position corresponding to amino acid        position 71 in SEQ ID NO: 1;    -   o) a threonine (T) at a position corresponding to amino acid        position 72 in SEQ ID NO: 1;    -   p) an alanine (A) at a position corresponding to amino acid        position 73 in SEQ ID NO: 1;    -   q) an arginine (R) at a position corresponding to amino acid        position 80 in SEQ ID NO: 1;    -   r) a lysine (K) at a position corresponding to amino acid        position 81 in SEQ ID NO: 1;    -   s) an arginine (R) at a position corresponding to amino acid        position 82 in SEQ ID NO: 1;    -   t) an aspartic acid (D) at a position corresponding to amino        acid position 86 in SEQ ID NO: 1;    -   u) an isoleucine (I) at a position corresponding to amino acid        position 91 in SEQ ID NO: 1;    -   v) an isoleucine (I) at a position corresponding to amino acid        position 95 in SEQ ID NO: 1;    -   w) an arginine (R) at a position corresponding to amino acid        position 98 in SEQ ID NO: 1;    -   x) a valine (V) at a position corresponding to amino acid        position 103 in SEQ ID NO: 1;    -   y) a serine (S) at a position corresponding to amino acid        position 113 in SEQ ID NO: 1;    -   z) a proline (P) at a position corresponding to amino acid        position 114 in SEQ ID NO: 1;    -   aa) an arginine (R) at a position corresponding to amino acid        position 116 in SEQ ID NO: 1;    -   bb) a glycine (G) at a position corresponding to amino acid        position 117 in SEQ ID NO: 1;    -   cc) a threonine (T) at a position corresponding to amino acid        position 118 in SEQ ID NO: 1;    -   dd) a an glycine (G) at a position corresponding to amino acid        position 121 in SEQ ID NO: 1;    -   ee) an arginine (R) at a position corresponding to amino acid        position 124 in SEQ ID NO: 1;    -   ff) a cysteine (C) at a position corresponding to amino acid        position 128 in SEQ ID NO: 1;    -   gg) an alanine (A) at a position corresponding to amino acid        position 129 in SEQ ID NO: 1;    -   hh) an arginine (R) at a position corresponding to amino acid        position 131 in SEQ ID NO: 1;    -   ii) a serine (S) at a position corresponding to amino acid        position 147 in SEQ ID NO: 1;    -   jj) an alanine (A) at a position corresponding to amino acid        position 151 in SEQ ID NO: 1;    -   kk) a leucine (L) or a methionine (M) at a position        corresponding to amino acid position 153 in SEQ ID NO: 1;    -   ll) a tryptophan (W) at a position corresponding to amino acid        position 159 in SEQ ID NO: 1;    -   mm) a glutamic acid (E) at a position corresponding to amino        acid position 160 in SEQ ID NO: 1;    -   nn) a valine (V) at a position corresponding to amino acid        position 161 in SEQ ID NO: 1;    -   oo) a tyrosine (Y) at a position corresponding to amino acid        position 162 in SEQ ID NO: 1;    -   pp) an arginine (R) at a position corresponding to amino acid        position 163 in SEQ ID NO: 1;    -   qq) a histidine (H) at a position corresponding to amino acid        position 164 in SEQ ID NO: 1;    -   rr) a leucine (L) at a position corresponding to amino acid        position 165 in SEQ ID NO: 1;    -   ss) an arginine (R) at a position corresponding to amino acid        position 166 in SEQ ID NO: 1;    -   tt) a histidine (H) at a position corresponding to amino acid        position 167 in SEQ ID NO: 1;    -   uu) a proline (P) at a position corresponding to amino acid        position 168 in SEQ ID NO: 1;    -   vv) an alanine (A) at a position corresponding to amino acid        position 169 in SEQ ID NO: 1;    -   ww) a proline (P) at a position corresponding to amino acid        position 170 in SEQ ID NO: 1;    -   xx) a histidine (H) at a position corresponding to amino acid        position 171 in SEQ ID NO: 1;    -   yy) a proline (P) at a position corresponding to amino acid        position 172 in SEQ ID NO: 1;    -   zz) an arginine (R) at a position corresponding to amino acid        position 173 in SEQ ID NO: 1;    -   aaa) a leucine (L) at a position corresponding to amino acid        position 174 in SEQ ID NO: 1;    -   bbb) a proline (P) at a position corresponding to amino acid        position 175 in SEQ ID NO: 1;    -   ccc) a glutamine (Q) at a position corresponding to amino acid        position 176 in SEQ ID NO: 1;    -   ddd) an alanine (A) at a position corresponding to amino acid        position 177 in SEQ ID NO: 1;    -   eee) an arginine (R) at a position corresponding to amino acid        position 178 in SEQ ID NO: 1;    -   fff) a valine (V) at a position corresponding to amino acid        position 179 in SEQ ID NO: 1;    -   ggg) a glutamine (Q) at a position corresponding to amino acid        position 180 in SEQ ID NO: 1;    -   hhh) a valine (V) at a position corresponding to amino acid        position 182 in SEQ ID NO: 1;    -   iii) a proline (P) at a position corresponding to amino acid        position 183 in SEQ ID NO: 1;    -   jjj) a lysine (K) at a position corresponding to amino acid        position 184 in SEQ ID NO: 1;    -   kkk) a threonine (T) or a histidine (H) at a position        corresponding to amino acid position 185 in SEQ ID NO: 1;    -   lll) a serine (S) at a position corresponding to amino acid        position 186 in SEQ ID NO: 1;    -   mmm) a glutamic acid (E) at a position corresponding to amino        acid position 187 in SEQ ID NO: 1;    -   nnn) a leucine (L) at a position corresponding to amino acid        position 188 in SEQ ID NO: 1;    -   ooo) a glutamic acid (E) at a position corresponding to amino        acid position 189 in SEQ ID NO: 1;    -   ppp) a glutamine (Q) at a position corresponding to amino acid        position 190 in SEQ ID NO: 1;    -   qqq) a leucine (L) at a position corresponding to amino acid        position 191 in SEQ ID NO: 1;    -   rrr) a proline (P) at a position corresponding to amino acid        position 194 in SEQ ID NO: 1;    -   sss) a lysine (K) at a position corresponding to amino acid        position 195 in SEQ ID NO: 1;    -   ttt) a serine (S) at a position corresponding to amino acid        position 196 in SEQ ID NO: 1;    -   uuu) a phenylalanine (F) at a position corresponding to amino        acid position 197 in SEQ ID NO: 1;    -   vvv) an isoleucine (I) at a position corresponding to amino acid        position 200 in SEQ ID NO: 1;    -   www) a valine (V) at a position corresponding to amino acid        position 203 in SEQ ID NO: 1;    -   xxx) a leucine (L) at a position corresponding to amino acid        position 204 in SEQ ID NO: 1;    -   yyy) a cysteine (C) at a position corresponding to amino acid        position 209 in SEQ ID NO: 1;    -   zzz) a leucine (L) at a position corresponding to amino acid        position 222 in SEQ ID NO: 1;    -   aaaa) an isoleucine (I) at a position corresponding to amino        acid position 232 in SEQ ID NO: 1;    -   bbbb) a serine (S) at a position corresponding to amino acid        position 236 in SEQ ID NO: 1;    -   cccc) a leucine (L) or an arginine (R) at a position        corresponding to amino acid position 237 in SEQ ID NO: 1;    -   dddd) a histidine (H) at a position corresponding to amino acid        position 246 in SEQ ID NO: 1;    -   eeee) an isoleucine (I) at a position corresponding to amino        acid position 254 in SEQ ID NO: 1;    -   ffff) a serine (S) at a position corresponding to amino acid        position 258 in SEQ ID NO: 1;    -   gggg) an arginine (R) at a position corresponding to amino acid        position 267 in SEQ ID NO: 1;    -   hhhh) an isoleucine (I) at a position corresponding to amino        acid position 278 in SEQ ID NO: 1;    -   iiii) a tyrosine (Y) at a position corresponding to amino acid        position 281 in SEQ ID NO: 1;    -   jjjj) a phenylalanine (F) at a position corresponding to amino        acid position 282 in SEQ ID NO: 1;    -   kkkk) a threonine (T) at a position corresponding to amino acid        position 289 in SEQ ID NO: 1;    -   llll) a glycine (G) at a position corresponding to amino acid        position 308 in SEQ ID NO: 1;    -   mmmm) an arginine (R) at a position corresponding to amino acid        position 311 in SEQ ID NO: 1;    -   nnnn) an alanine (A) at a position corresponding to amino acid        position 312 in SEQ ID NO: 1;    -   oooo) an alanine (A) at a position corresponding to amino acid        position 316 in SEQ ID NO: 1;    -   pppp) an arginine (R) at a position corresponding to amino acid        position 318 in SEQ ID NO: 1    -   qqqq) an alanine (A) at a position corresponding to amino acid        position 334 in SEQ ID NO: 1;    -   rrrr) a phenylalanine (F) at a position corresponding to amino        acid position 339 in SEQ ID NO: 1;    -   ssss) a glycine (G) or a leucine (L) at a position corresponding        to amino acid position 340 in SEQ ID NO: 1;    -   tttt) a serine (S) at a position corresponding to amino acid        position 342 in SEQ ID NO: 1;    -   uuuu) an asparagine (N) at a position corresponding to amino        acid position 345 in SEQ ID NO: 1;    -   vvvv) an asparagine (N) at a position corresponding to amino        acid position 346 in SEQ ID NO: 1;    -   wwww) an asparagine (N) at a position corresponding to amino        acid position 348 in SEQ ID NO: 1; or,    -   xxxx) any combination of a) to wwww).-   32. The isolated polypeptide of embodiment 29, wherein said    polypeptide further comprises:    -   a) an aspartic acid (D) at a position corresponding to amino        acid position 2 in SEQ ID NO: 1;    -   b) a histidine (H) at a position corresponding to amino acid        position 12 in SEQ ID NO: 1;    -   c) an isoleucine (I) at a position corresponding to amino acid        position 16 in SEQ ID NO: 1;    -   d) a serine (S) or an alanine (A) at a position corresponding to        amino acid position 19 in SEQ ID NO: 1;    -   e) a cysteine (C) at a position corresponding to amino acid        position 22 in SEQ ID NO: 1;    -   f) a leucine (L) at a position corresponding to amino acid        position 23 in SEQ ID NO: 1;    -   g) a methionine (M) at a position corresponding to amino acid        position 24 in SEQ ID NO: 1;    -   h) an arginine (R) or an alanine (A) at a position corresponding        to amino acid position 28 in SEQ ID NO: 1;    -   i) an arginine (R), alanine (A), glutamine (Q), cysteine (C),        glycine (G), serine (S), threonine (T), leucine (L), glutamic        acid (E), or a proline (P) at a position corresponding to amino        acid position 30 in SEQ ID NO: 1;    -   j) an arginine (R) at a position corresponding to amino acid        position 31 in SEQ ID NO: 1;    -   k) an arginine (R), alanine (A), lysine (K) glutamine (Q),        glycine (G) or a leucine (L) at a position corresponding to        amino acid position 32 in SEQ ID NO: 1;    -   l) an asparagine (N) at a position corresponding to amino acid        position 36 in SEQ ID NO: 1;    -   m) a leucine (L) at a position corresponding to amino acid        position 43 in SEQ ID NO: 1;    -   n) an arginine (R) or lysine (K) at a position corresponding to        amino acid position 50 in SEQ ID NO: 1;    -   o) an isoleucine (I) or a leucine (L) at a position        corresponding to amino acid position 54 in SEQ ID NO: 1;    -   p) a leucine (L) at a position corresponding to amino acid        position 56 in SEQ ID NO: 1;    -   q) a glutamic acid (E) at a position corresponding to amino acid        position 57 in SEQ ID NO: 1;    -   r) an isoleucine (I) at a position corresponding to amino acid        position 58 in SEQ ID NO: 1;    -   s) a histidine (H) or alanine (A) at a position corresponding to        amino acid position 59 in SEQ ID NO: 1;    -   t) a valine (V) at a position corresponding to amino acid        position 62 in SEQ ID NO: 1;    -   u) a lysine (K) at a position corresponding to amino acid        position 71 in SEQ ID NO: 1;    -   v) a threonine (T) at a position corresponding to amino acid        position 72 in SEQ ID NO: 1;    -   w) an alanine (A) at a position corresponding to amino acid        position 73 in SEQ ID NO: 1;    -   x) a glycine (G) at a position corresponding to amino acid        position 79 in SEQ ID NO: 1;    -   y) an arginine (R) at a position corresponding to amino acid        position 80 in SEQ ID NO: 1;    -   z) a lysine (K) at a position corresponding to amino acid        position 81 in SEQ ID NO: 1;    -   aa) an arginine (R) at a position corresponding to amino acid        position 82 in SEQ ID NO: 1;    -   bb) an aspartic acid (D) at a position corresponding to amino        acid position 86 in SEQ ID NO: 1;    -   cc) a leucine (L) at a position corresponding to amino acid        position 87 in SEQ ID NO: 1;    -   dd) an isoleucine (I) at a position corresponding to amino acid        position 91 in SEQ ID NO: 1;    -   ee) an isoleucine (I) at a position corresponding to amino acid        position 95 in SEQ ID NO: 1;    -   ff) an arginine (R) at a position corresponding to amino acid        position 98 in SEQ ID NO: 1;    -   gg) a valine (V) at a position corresponding to amino acid        position 103 in SEQ ID NO: 1;    -   hh) an alanine (A) at a position corresponding to amino acid        position 105 in SEQ ID NO: 1;    -   ii) an arginine (R) at a position corresponding to amino acid        position 111 in SEQ ID NO: 1;    -   jj) a serine (S) at a position corresponding to amino acid        position 113 in SEQ ID NO: 1;    -   kk) a proline (P) at a position corresponding to amino acid        position 114 in SEQ ID NO: 1;    -   ll) an arginine (R) at a position corresponding to amino acid        position 116 in SEQ ID NO: 1;    -   mm) a an glycine (G) at a position corresponding to amino acid        position 117 in SEQ ID NO: 1;    -   nn) a threonine (T) at a position corresponding to amino acid        position 118 in SEQ ID NO: 1;    -   oo) a an glycine (G) at a position corresponding to amino acid        position 121 in SEQ ID NO: 1;    -   pp) an arginine (R) at a position corresponding to amino acid        position 124 in SEQ ID NO: 1;    -   qq) a cysteine (C) at a position corresponding to amino acid        position 128 in SEQ ID NO: 1;    -   rr) an alanine (A) at a position corresponding to amino acid        position 129 in SEQ ID NO: 1;    -   ss) an arginine (R) at a position corresponding to amino acid        position 131 in SEQ ID NO: 1;    -   tt) a valine (V) at a position corresponding to amino acid        position 132 in SEQ ID NO: 1;    -   uu) a serine (S) at a position corresponding to amino acid        position 147 in SEQ ID NO: 1;    -   vv) an alanine (A) at a position corresponding to amino acid        position 151 in SEQ ID NO: 1;    -   ww) a leucine (L) or a methionine (M) at a position        corresponding to amino acid position 153 in SEQ ID NO: 1;    -   xx) a tryptophan (W) at a position corresponding to amino acid        position 159 in SEQ ID NO: 1;    -   yy) a glutamic acid (E) at a position corresponding to amino        acid position 160 in SEQ ID NO: 1;    -   zz) a valine (V) at a position corresponding to amino acid        position 161 in SEQ ID NO: 1;    -   aaa) a tyrosine (Y) at a position corresponding to amino acid        position 162 in SEQ ID NO: 1;    -   bbb) an arginine (R) at a position corresponding to amino acid        position 163 in SEQ ID NO: 1;    -   ccc) a histidine (H) at a position corresponding to amino acid        position 164 in SEQ ID NO: 1;    -   ddd) a leucine (L) at a position corresponding to amino acid        position 165 in SEQ ID NO: 1;    -   eee) an arginine (R) at a position corresponding to amino acid        position 166 in SEQ ID NO: 1;    -   fff) a histidine (H) at a position corresponding to amino acid        position 167 in SEQ ID NO: 1;    -   ggg) a proline (P) at a position corresponding to amino acid        position 168 in SEQ ID NO: 1;    -   hhh) an alanine (A) at a position corresponding to amino acid        position 169 in SEQ ID NO: 1;    -   iii) a proline (P) at a position corresponding to amino acid        position 170 in SEQ ID NO: 1;    -   jjj) a histidine (H) at a position corresponding to amino acid        position 171 in SEQ ID NO: 1;    -   kkk) a proline (P) at a position corresponding to amino acid        position 172 in SEQ ID NO: 1;    -   lll) an arginine (R) at a position corresponding to amino acid        position 173 in SEQ ID NO: 1;    -   mmm) a leucine (L) at a position corresponding to amino acid        position 174 in SEQ ID NO: 1;    -   nnn) a proline (P) at a position corresponding to amino acid        position 175 in SEQ ID NO: 1;    -   ooo) a glutamine (Q) at a position corresponding to amino acid        position 176 in SEQ ID NO: 1;    -   ppp) an alanine (A) at a position corresponding to amino acid        position 177 in SEQ ID NO: 1;    -   qqq) an arginine (R) at a position corresponding to amino acid        position 178 in SEQ ID NO: 1;    -   rrr) a valine (V) at a position corresponding to amino acid        position 179 in SEQ ID NO: 1;    -   sss) a glutamine (Q) at a position corresponding to amino acid        position 180 in SEQ ID NO: 1;    -   ttt) a valine (V) at a position corresponding to amino acid        position 182 in SEQ ID NO: 1;    -   uuu) a proline (P) at a position corresponding to amino acid        position 183 in SEQ ID NO: 1;    -   vvv) a lysine (K) at a position corresponding to amino acid        position 184 in SEQ ID NO: 1;    -   www) a threonine (T) or a histidine (H) at a position        corresponding to amino acid position 185 in SEQ ID NO: 1;    -   xxx) a serine (S) at a position corresponding to amino acid        position 186 in SEQ ID NO: 1;    -   yyy) a glutamic acid (E) at a position corresponding to amino        acid position 187 in SEQ ID NO: 1;    -   zzz) a leucine (L) at a position corresponding to amino acid        position 188 in SEQ ID NO: 1;    -   aaaa) a glutamic acid (E) at a position corresponding to amino        acid position 189 in SEQ ID NO: 1;    -   bbbb) a glutamine (Q) at a position corresponding to amino acid        position 190 in SEQ ID NO: 1;    -   cccc) a leucine (L) at a position corresponding to amino acid        position 191 in SEQ ID NO: 1;    -   dddd) an amino acid deletion at a position corresponding to        amino acid position 192 in SEQ ID NO: 1;    -   eeee) a proline (P) at a position corresponding to amino acid        position 194 in SEQ ID NO: 1;    -   ffff) a lysine (K) at a position corresponding to amino acid        position 195 in SEQ ID NO: 1;    -   gggg) a serine (S) at a position corresponding to amino acid        position 196 in SEQ ID NO: 1;    -   hhhh) a phenylalanine (F) at a position corresponding to amino        acid position 197 in SEQ ID NO: 1;    -   iiii) an isoleucine (I) at a position corresponding to amino        acid position 200 in SEQ ID NO: 1;    -   jjjj) a valine (V) at a position corresponding to amino acid        position 203 in SEQ ID NO: 1;    -   kkkk) a leucine (L) at a position corresponding to amino acid        position 204 in SEQ ID NO: 1;    -   llll) an alanine (A) or a serine (S) at a position corresponding        to amino acid position 206 in SEQ ID NO: 1;    -   mmmm) a cysteine (C) at a position corresponding to amino acid        position 209 in SEQ ID NO: 1;    -   nnnn) a leucine (L) at a position corresponding to amino acid        position 222 in SEQ ID NO: 1;    -   oooo) a methionine (M) at a position corresponding to amino acid        position 211 in SEQ ID NO: 1;    -   pppp) an isoleucine (I) at a position corresponding to amino        acid position 232 in SEQ ID NO: 1;    -   qqqq) a serine (S) at a position corresponding to amino acid        position 236 in SEQ ID NO: 1;    -   rrrr) a leucine (L) or an arginine (R) at a position        corresponding to amino acid position 237 in SEQ ID NO: 1;    -   ssss) an isoleucine (I) or a leucine (L) at a position        corresponding to amino acid position 241 in SEQ ID NO: 1;    -   tttt) a glutamic acid (E) at a position corresponding to amino        acid position 244 in SEQ ID NO: 1;    -   uuuu) a histidine (H) at a position corresponding to amino acid        position 246 in SEQ ID NO: 1;    -   vvvv) an aspartic acid (D) or histidine (H) at a position        corresponding to amino acid position 253 in SEQ ID NO: 1;    -   wwww) an isoleucine (I) at a position corresponding to amino        acid position 254 in SEQ ID NO: 1;    -   xxxx) a serine (S) at a position corresponding to amino acid        position 258 in SEQ ID NO: 1;    -   yyyy) an arginine (R) at a position corresponding to amino acid        position 267 in SEQ ID NO: 1;    -   zzzz) an isoleucine (I) at a position corresponding to amino        acid position 278 in SEQ ID NO: 1;    -   aaaaa) a tyrosine (Y) at a position corresponding to amino acid        position 281 in SEQ ID NO: 1;    -   bbbbb) a phenylalanine (F) at a position corresponding to amino        acid position 282 in SEQ ID NO: 1;    -   ccccc) a threonine (T) at a position corresponding to amino acid        position 289 in SEQ ID NO: 1;    -   ddddd) an alanine (A) at a position corresponding to amino acid        position 292 in SEQ ID NO: 1;    -   eeeee) a glycine (G) at a position corresponding to amino acid        position 308 in SEQ ID NO: 1;    -   fffff) an arginine (R) at a position corresponding to amino acid        position 311 in SEQ ID NO: 1;    -   ggggg) an alanine (A) at a position corresponding to amino acid        position 312 in SEQ ID NO: 1;    -   hhhhh) an alanine (A) at a position corresponding to amino acid        position 316 in SEQ ID NO: 1;    -   iiiii) an arginine (R) at a position corresponding to amino acid        position 318 in SEQ ID NO: 1    -   jjjjj) a valine (V) at a position corresponding to amino acid        position 319 in SEQ ID NO: 1;    -   kkkkk) an alanine (A) at a position corresponding to amino acid        position 334 in SEQ ID NO: 1;    -   lllll) a phenylalanine (F) at a position corresponding to amino        acid position 339 in SEQ ID NO: 1;    -   mmmmm) a glycine (G) or a leucine (L) at a position        corresponding to amino acid position 340 in SEQ ID NO: 1;    -   nnnnn) a serine (S) at a position corresponding to amino acid        position 342 in SEQ ID NO: 1;    -   ooooo) an asparagine (N) at a position corresponding to amino        acid position 345 in SEQ ID NO: 1;    -   ppppp) an asparagine (N) at a position corresponding to amino        acid position 346 in SEQ ID NO: 1; or,    -   qqqqq) an asparagine (N) at a position corresponding to amino        acid position 348 in SEQ ID NO: 1; or,    -   rrrrr) any combination of a) to qqqqq).-   33. The isolated polypeptide of embodiment 29 selected from the    group consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22,    23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 87,    88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,    104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,    117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,    130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,    143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,    156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 251,    252, 253, 272, 273, 274, 275, 272, 273, 274, 275, 284, 285, 286,    287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 330,    331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 357, 358,    359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371,    390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402,    430, 431, 432 and 433.-   34. The isolated polypeptide of embodiment 29, wherein the    polypeptide is capable of recognizing and cleaving a meganuclease    recognition sites selected from the group consisting of SEQ ID NO: 2    (LIG3-4), SEQ ID NO: 85 (MHP77), SEQ ID NO: 269 (MS26), SEQ ID NO:    281 (MHP14), SEQ ID NO: 331(MP107), SEQ ID NO: 358 (ZM6.3), SEQ ID    NO: 390 (ZM6.22v2), SEQ ID NO: 423 or SEQ ID NO: 424.-   35. The isolated polypeptide of embodiment 29, wherein said    polypeptide has an increased meganuclease activity when compared to    a control meganuclease that lacks said amino acid modification.-   36. The isolated polypeptide of embodiment 29, wherein said control    meganuclease is selected from the group of SEQ ID NO: 1 (LIG3-4),    SEQ ID NO: 86 (MHP77), SEQ ID NO: 250 (MHP77.3), SEQ ID NO: 270    (MS26+), SEQ ID NO: 271, SEQ ID NO: 282 (MHP14), SEQ ID NO: 283    (MHP14+), SEQ ID NO: 329 (MP107), SEQ ID NO: 356 (ZM6.3), SEQ ID NO:    389 (ZM6.22v2), SEQ ID NO: 429 or SEQ ID NO: 435.-   37. The isolated polypeptide of embodiment 29, wherein the increased    meganuclease activity is evidenced by:    -   a) a higher yeast assay score when compared to the control        meganuclease that lacks said amino acid modification; or,    -   b) a higher target site mutation rate when compared to the        control meganuclease that lacks said amino acid modification;        or,    -   c) a higher in-vitro cutting when compared to the control        meganuclease that lacks said amino acid modification; or,    -   d) any combination of (a), (b) and (c).-   38. A composition comprising at least one or more polypeptides of    embodiment 29.-   39. A method for producing a meganuclease having increased activity    over a range of temperatures, the method comprising:    -   a) producing a variant meganuclease by modifying at least one        amino acid at an amino acid position corresponding to a position        of SEQ ID NO: 1 selected from the group consisting of positions        2, 12, 16, 22, 23, 31, 36, 43, 50, 56, 58, 59, 62, 71, 72, 73,        80, 81, 82, 86, 91, 95, 98, 103, 113, 114, 116, 117, 118, 121,        124, 128, 129, 131, 147, 151, 153, 159, 160, 161, 162, 163, 164,        165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,        178, 179, 180, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,        192, 194, 195, 196, 197, 200, 203, 204, 209, 222, 232, 236, 237,        246, 254, 258, 267, 278, 281, 282, 289, 308, 311, 312, 316, 318,        319, 334, 339, 340, 342, 345, 346 348 and combinations thereof;        and,    -   b) selecting said variant meganuclease from step a) and        screening said variant meganuclease for the ability to cleave a        DNA target sequence over a range of temperatures between and        including 16° C. to 37° C.-   40. The method of embodiment 39, wherein said range of temperatures    comprises:    -   a) 16° C.;    -   b) 18° C.;    -   c) 20° C.;    -   d) 24° C.;    -   e) 28° C.;    -   f) 30° C.;    -   g) 37° C.; or,    -   h) any combination of a), b), c), d), e), f), h), g) and g).-   41. A method for producing a meganuclease having an increased    meganuclease activity when compared to a control meganuclease, the    method comprising:    -   a) producing a variant meganuclease by modifying at least one        amino acid at an amino acid position corresponding to a position        of SEQ ID NO: 1 selected from the group consisting of positions        2, 12, 16, 22, 23, 31, 36, 43, 50, 56, 58, 59, 62, 71, 72, 73,        80, 81, 82, 86, 91, 95, 98, 103, 113, 114, 116, 117, 118, 121,        124, 128, 129, 131, 147, 151, 153, 159, 160, 161, 162, 163, 164,        165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,        178, 179, 180, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,        192, 194, 195, 196, 197, 200, 203, 204, 209, 222, 232, 236, 237,        246, 254, 258, 267, 278, 281, 282, 289, 308, 311, 312, 316, 318,        319, 334, 339, 340, 342, 345, 346, 348 and combinations thereof;        and,    -   b) selecting the variant meganuclease from step a) and screening        said variant for increased meganuclease activity when compared        to a control meganuclease.-   42. The method of embodiment 41, wherein the increased meganuclease    activity is evidenced by:    -   a) a higher yeast assay score when compared to the control        meganuclease that lacks said amino acid modification; or,    -   b) a higher target site mutation rate when compared to the        control meganuclease that lacks said amino acid modification;        or,    -   c) a higher in-vitro cutting when compared to the control        meganuclease that lacks said amino acid modification; or,    -   d) any combination of (a), (b) and (c).-   43. The isolated or recombinant polynucleotide of embodiment 1,    wherein said meganuclease polypeptide comprises a linker    polypeptide, wherein said linker polypeptide comprises:    -   a) SEQ ID NO: 420;    -   b) SEQ ID NO: 421;    -   c) SEQ ID NO: 422; or,    -   d) an amino acid sequence consisting of any possible amino acid        at positions corresponding to positions 156 to 193 of SEQ ID NO:        1.-   44. A composition comprising at least one or more polynucleotides of    embodiment 1.-   45. An isolated or recombinant polynucleotide encoding a    meganuclease polypeptide, said polypeptide comprising an amino acid    sequence having at least one amino acid modification at an amino    acid position corresponding to a position of SEQ ID NO: 1 selected    from the group consisting of positions 16, 22, 50, 56, 59, 71, 81,    103, 121, 153, 185, 209, 222, 246, 258, 281, 308, 316, 345, 346, and    combinations thereof, and wherein the polypeptide is capable of    recognizing and cleaving a meganuclease target site comprising SEQ    ID NO: 2.-   46. The isolated or recombinant polynucleotide of embodiment 45,    wherein said nucleotide sequence encodes a meganuclease polypeptide    having at least 80% sequence identity to SEQ ID NO: 1.-   47. The isolated or recombinant polynucleotide of embodiment 45,    wherein said at least one amino acid modification comprises any one    of the amino acid modifications shown in FIG. 5A-FIG. 5E.-   48. The isolated or recombinant polynucleotide of embodiment 45,    wherein said nucleotide sequence encodes a meganuclease polypeptide    selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17,    18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,    35, 36, 37 and 38.-   49. An isolated or recombinant polynucleotide encoding a    meganuclease polypeptide, the polypeptide comprising an amino acid    sequence having at least one amino acid modification at an amino    acid position corresponding to a position of SEQ ID NO: 86 selected    from the group consisting of positions 2, 12, 16, 22, 23, 36, 43,    50, 56, 58, 59, 72, 73, 81, 86, 91, 95, 103, 113, 114, 120, 121,    124, 128, 129, 131, 151, 153, 200, 204, 209, 232, 236, 237, 246,    254, 258, 267, 281, 308, 311, 312, 316, 319, 334, 339, 340, 342, and    combinations thereof, and wherein the polypeptide is capable of    recognizing and cleaving a meganuclease target site comprising SEQ    ID NO: 85.-   50. The isolated or recombinant polynucleotide of embodiment 49,    wherein said nucleotide sequence encodes a meganuclease polypeptide    having at least 80% sequence identity to SEQ ID NO: 86.-   51. The isolated or recombinant polynucleotide of embodiment 49,    wherein said at least one amino acid modification comprises any one    of the amino acid modifications shown in FIG. 9A-FIG. 9N.-   52. The isolated or recombinant polynucleotide of embodiment 49,    wherein said nucleotide sequence encodes a meganuclease polypeptide    selected from the group consisting of SEQ ID NOs: 87, 88, 89, 90,    91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,    106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,    119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,    132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,    145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,    158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 251, 252 and 253.-   53. An isolated or recombinant polynucleotide encoding a    meganuclease polypeptide, the polypeptide comprising an amino acid    sequence having at least one amino acid modification at an amino    acid position corresponding to a position of SEQ ID NO: 270 selected    from the group consisting of positions 16, 22, 50, 71, 185, 246,    258, 316 and combinations thereof, and wherein the polypeptide is    capable of recognizing and cleaving a meganuclease target site    comprising SEQ ID NO: 269.-   54. The isolated or recombinant polynucleotide of embodiment 53,    wherein said nucleotide sequence encodes a meganuclease polypeptide    selected from the group consisting of SEQ ID NOs: 272, 273, 274 and    275.-   55. An isolated or recombinant polynucleotide encoding a    meganuclease polypeptide, the polypeptide comprising an amino acid    sequence having at least one amino acid modification at an amino    acid position corresponding to a position of SEQ ID NO: 282 selected    from the group consisting of positions 12, 16, 22, 31, 50, 56, 59,    62, 81, 98, 103, 105, 116, 118, 121, 153, 159, 160, 161, 162, 163,    164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,    177, 178, 179, 180, 182, 183, 184, 185, 186, 187, 188, 189, 190,    191, 192, 258, 281, 308, 312, 316, 319, and combinations thereof,    and wherein the polypeptide is capable of recognizing and cleaving a    meganuclease target site comprising SEQ ID NO: 281.-   56. The isolated or recombinant polynucleotide of embodiment 55,    wherein said nucleotide sequence encodes a meganuclease polypeptide    having at least 80% sequence identity to SEQ ID NO: 282.-   57. The isolated or recombinant polynucleotide of embodiment 55,    wherein said at least one amino acid modification comprises any one    of the amino acid modifications shown in FIG. 10A-FIG. 10D.-   58. The isolated or recombinant polynucleotide of embodiment 55,    wherein said nucleotide sequence encodes a meganuclease polypeptide    selected from the group consisting of SEQ ID NOs: 284, 285, 286,    287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297 and 298.-   59. An isolated or recombinant polynucleotide encoding a    meganuclease polypeptide, the polypeptide comprising an amino acid    sequence having at least one amino acid modification at an amino    acid position corresponding to a position of SEQ ID NO: 329 selected    from the group consisting of positions 12, 32, 50, 56, 80, 105, 124,    129, 131, 153, 185, 311, 316, 318, 340, and combinations thereof,    and wherein the polypeptide is capable of recognizing and cleaving a    meganuclease target site comprising SEQ ID NO: 328.-   60. The isolated or recombinant polynucleotide of embodiment 59,    wherein said nucleotide sequence encodes a meganuclease polypeptide    having at least 80% sequence identity to SEQ ID NO: 329.-   61. The isolated or recombinant polynucleotide of embodiment 59,    wherein said at least one amino acid modification comprises any one    of the amino acid modifications shown in FIG. 11.-   62. The isolated or recombinant polynucleotide of embodiment 59,    wherein said nucleotide sequence encodes a meganuclease polypeptide    selected from the group consisting of SEQ ID NOs: 330, 331, 332,    333, 334, 335, 336, 337, 338, 339, 340 and 341.-   63. An isolated or recombinant polynucleotide encoding a    meganuclease polypeptide, the polypeptide comprising an amino acid    sequence having at least one amino acid modification at an amino    acid position corresponding to a position of SEQ ID NO: 356 selected    from the group consisting of positions 12, 24, 36, 50, 56, 62, 73,    80, 124, 129, 147, 182, 203, 237, 252, 311, 316, 318, 340, 348, and    combinations thereof, and wherein the polypeptide is capable of    recognizing and cleaving a meganuclease target site comprising SEQ    ID NO: 355.    64. The isolated or recombinant polynucleotide of embodiment 63,    wherein said nucleotide sequence encodes a meganuclease polypeptide    having at least 80% sequence identity to SEQ ID NO: 356.-   65. The isolated or recombinant polynucleotide of embodiment 63,    wherein said at least one amino acid modification comprises any one    of the amino acid modifications shown in FIG. 12.-   66. The isolated or recombinant polynucleotide of embodiment 63,    wherein said nucleotide sequence encodes a meganuclease polypeptide    selected from the group consisting of SEQ ID NOs: 357, 358, 359,    360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, and 371.-   67. An isolated or recombinant polynucleotide encoding a    meganuclease polypeptide, the polypeptide comprising an amino acid    sequence having at least one amino acid modification at an amino    acid position corresponding to a position of SEQ ID NO: 389 selected    from the group consisting of positions 12, 50, 56, 124, 129, 131,    153, 211, 237, 311, 316, and position 318, and combinations thereof,    and wherein the polypeptide is capable of recognizing and cleaving a    meganuclease target site comprising SEQ ID NO: 388.-   68. The isolated or recombinant polynucleotide of embodiment 67,    wherein said nucleotide sequence encodes a meganuclease polypeptide    having at least 80% sequence identity to SEQ ID NO: 389.-   69. The isolated or recombinant polynucleotide of embodiment 67,    wherein said at least one amino acid modification comprises any one    of the amino acid modifications shown in FIG. 13.-   70. The isolated or recombinant polynucleotide of embodiment 67,    wherein said nucleotide sequence encodes a meganuclease polypeptide    selected from the group consisting of SEQ ID NOs: 390, 391, 392,    393, 394, 395, 396, 397, 398, 399, 400, 401, 402, and 403.-   72. A yeast, plant, plant cell, explant or seed comprising the    meganuclease created by the method of embodiments 36-42.-   73. A method of introducing a double-strand break in the genome of a    yeast or plant cell, said method comprising:    -   a) contacting at least one plant or yeast cell comprising in its        genome a meganuclease recognition site with a variant        meganuclease polypeptide selected from the group consisting of        SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 87, 88, 89, 90,        91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,        105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,        118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,        131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,        144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,        157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 251, 252,        253, 272, 273, 274, 275, 272, 273, 274, 275, 284, 285, 286, 287,        288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 330, 331,        332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 357, 358, 359,        360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 390,        391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402 and        403, wherein the variant meganuclease is capable of inducing a        double-strand break in said recognition site; and,    -   b) selecting the yeast or plant cell from a) and screening said        yeast or plant cell for any modification of said recognition        sequence.-   74. A method of integrating a polynucleotide of interest into a    recognition site in the genome of a plant or yeast cell, the method    comprising:    -   a) contacting at least one plant or yeast cell comprising in its        genome a meganuclease recognition site with:    -   (i) a variant meganuclease polypeptide selected from the group        consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22,        23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,        87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,        102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,        115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,        128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,        141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,        154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,        167, 251, 252, 253, 272, 273, 274, 275, 272, 273, 274, 275, 284,        285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,        298, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341,        357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369,        370, 371, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400,        401, 402 and 403, wherein the variant meganuclease is capable of        inducing a double-strand break in said recognition site; and,    -   (ii) a DNA fragment containing a polynucleotide of interest;    -   b) selecting at least one plant or yeast cell comprising        integration of the polynucleotide of interest cassette at the        recognition site.-   75. An isolated or recombinant polynucleotide encoding a    meganuclease polypeptide, the polypeptide comprising an amino acid    sequence having at least one amino acid modification at an amino    acid position corresponding to a position of SEQ ID NO: 429 selected    from the group consisting of positions 16, 22, 50, 71, 185, 246,    258, 316 and combinations thereof, and wherein the polypeptide is    capable of recognizing and cleaving a meganuclease target site    comprising SEQ ID NO: 423.-   76. The isolated or recombinant polynucleotide of embodiment 75,    wherein said nucleotide sequence encodes a meganuclease polypeptide    selected from the group consisting of SEQ ID NOs: 430, 431 and 432.-   77. An isolated or recombinant polynucleotide encoding a    meganuclease polypeptide of SEQ ID NOs: 436, wherein the polypeptide    is capable of recognizing and cleaving a meganuclease target site    comprising SEQ ID NO: 424.

EXPERIMENTAL Example 1 Transformation of Maize Immature Embryos

Transformation can be accomplished by various methods known to beeffective in plants, including particle-mediated delivery,Agrobacterium-mediated transformation, PEG-mediated delivery, andelectroporation.

a. Particle-Mediated Delivery

Transformation of maize immature embryos using particle delivery isperformed as follows. Media recipes follow below.

The ears are husked and surface sterilized in 30% Clorox bleach plus0.5% Micro detergent for 20 minutes, and rinsed two times with sterilewater. The immature embryos are isolated and placed embryo axis sidedown (scutellum side up), 25 embryos per plate, on 560Y medium for 4hours and then aligned within the 2.5-cm target zone in preparation forbombardment. Alternatively, isolated embryos are placed on 560L(Initiation medium) and placed in the dark at temperatures ranging from26° C. to 37° C. for 8 to 24 hours prior to placing on 560Y for 4 hoursat 26° C. prior to bombardment as described above.

Plasmids containing the double strand brake inducing agent and donor DNAare constructed using standard molecular biology techniques andco-bombarded with plasmids containing the developmental genes ODP2 (AP2domain transcription factor ODP2 (Ovule development protein 2);US20090328252 A1) and Wushel (US2011/0167516).

The plasmids and DNA of interest are precipitated onto 0.6 μm (averagediameter) gold pellets using a water-soluble cationic lipid Tfx™-50(Cat# E1811, Promega, Madison, Wis., USA) as follows. DNA solution isprepared on ice using 1 □g of plasmid DNA and optionally otherconstructs for co-bombardment such as 50 ng (0.5 □l) of each plasmidcontaining the developmental genes ODP2 (AP2 domain transcription factorODP2 (Ovule development protein 2); US20090328252 A1) and Wushel. To thepre-mixed DNA, 20 μl of prepared gold particles (15 mg/ml) and 5 □lTfx-50 is added in water and mixed carefully. Gold particles arepelleted in a microfuge at 10,000 rpm for 1 min and supernatant isremoved. The resulting pellet is carefully rinsed with 100 ml of 100%EtOH without resuspending the pellet and the EtOH rinse is carefullyremoved. 105 □l of 100% EtOH is added and the particles are resuspendedby brief sonication. Then, 10 μl is spotted onto the center of eachmacrocarrier and allowed to dry about 2 minutes before bombardment.

Alternatively, the plasmids and DNA of interest are precipitated onto1.1 μm (average diameter) tungsten pellets using a calcium chloride(CaCl₂) precipitation procedure by mixing 100 μl prepared tungstenparticles in water, 10 μl (1 μg) DNA in Tris EDTA buffer (1 μg totalDNA), 100 μl 2.5M CaCl2, and 10 μl 0.1M spermidine. Each reagent isadded sequentially to the tungsten particle suspension, with mixing. Thefinal mixture is sonicated briefly and allowed to incubate underconstant vortexing for 10 minutes. After the precipitation period, thetubes are centrifuged briefly, liquid is removed, and the particles arewashed with 500 ml 100% ethanol, followed by a 30 second centrifugation.Again, the liquid is removed, and 105 μl 100% ethanol is added to thefinal tungsten particle pellet. For particle gun bombardment, thetungsten/DNA particles are briefly sonicated. 10 μl of the tungsten/DNAparticles is spotted onto the center of each macrocarrier, after whichthe spotted particles are allowed to dry about 2 minutes beforebombardment.

The sample plates are bombarded at level #4 with a Biorad Helium Gun.All samples receive a single shot at 450 PSI, with a total of tenaliquots taken from each tube of prepared particles/DNA.

Following bombardment, the embryos are incubated on 560P (maintenancemedium) for 12 to 48 hours at temperatures ranging from 26 C to 37 C,and then placed at 26 C. After 5 to 7 days the embryos are transferredto 560R selection medium containing 3 mg/liter Bialaphos, andsubcultured every 2 weeks at 26 C. After approximately 10 weeks ofselection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to a lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto a 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to Classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for transformation efficiency, and/ormodification of regenerative capabilities.

Initiation medium (560L) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/lthiamine HCl, 20.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline(brought to volume with D-I H2O following adjustment to pH 5.8 withKOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H2O); and8.5 mg/l silver nitrate (added after sterilizing the medium and coolingto room temperature).

Maintenance medium (560P) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/lthiamine HCl, 30.0 g/l sucrose, 2.0 mg/l 2,4-D, and 0.69 g/l L-proline(brought to volume with D-I H2O following adjustment to pH 5.8 withKOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H2O); and0.85 mg/l silver nitrate (added after sterilizing the medium and coolingto room temperature).

Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/lthiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline(brought to volume with D-I H2O following adjustment to pH 5.8 withKOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H2O); and8.5 mg/l silver nitrate (added after sterilizing the medium and coolingto room temperature).

Selection medium (560R) comprises 4.0 g/l N6 basal salts (SIGMA C-1416),1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/l thiamineHCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought to volume with D-IH2O following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (addedafter bringing to volume with D-I H2O); and 0.85 mg/l silver nitrate and3.0 mg/l bialaphos (both added after sterilizing the medium and coolingto room temperature).

Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycinebrought to volume with polished D-I H2O) (Murashige and Skoog (1962)Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/lsucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume withpolished D-I H2O after adjusting to pH 5.6); 3.0 g/l Gelrite (addedafter bringing to volume with D-I H2O); and 1.0 mg/l indoleacetic acidand 3.0 mg/l bialaphos (added after sterilizing the medium and coolingto 60° C.).

Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO 11117-074),5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/lthiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought tovolume with polished D-I H2O), 0.1 g/l myo-inositol, and 40.0 g/lsucrose (brought to volume with polished D-I H2O after adjusting pH to5.6); and 6 g/l bacto-agar (added after bringing to volume with polishedD-I H2O), sterilized and cooled to 60° C.

b. Agrobacterium-Mediated Transformation

Agrobacterium-mediated transformation was performed essentially asdescribed in Djukanovic et al. (2006) Plant Biotech J 4:345-57. Briefly,10-12 day old immature embryos (0.8-2.5 mm in size) were dissected fromsterilized kernels and placed into liquid medium (4.0 g/L N6 Basal Salts(Sigma C-1416), 1.0 ml/L Eriksson's Vitamin Mix (Sigma E-1511), 1.0 mg/Lthiamine HCl, 1.5 mg/L 2, 4-D, 0.690 g/L L-proline, 68.5 g/L sucrose,36.0 g/L glucose, pH 5.2). After embryo collection, the medium wasreplaced with 1 ml Agrobacterium at a concentration of 0.35-0.45 OD550.Maize embryos were incubated with Agrobacterium for 5 min at roomtemperature, then the mixture was poured onto a media plate containing4.0 g/L N6 Basal Salts (Sigma C-1416), 1.0 ml/L Eriksson's Vitamin Mix(Sigma E-1511), 1.0 mg/L thiamine HCl, 1.5 mg/L 2, 4-D, 0.690 g/LL-proline, 30.0 g/L sucrose, 0.85 mg/L silver nitrate, 0.1 nMacetosyringone, and 3.0 g/L Gelrite, pH 5.8. Embryos were incubated axisdown, in the dark for 3 days at 20° C., then incubated 4 days in thedark at 28° C., then transferred onto new media plates containing 4.0g/L N6 Basal Salts (Sigma C-1416), 1.0 ml/L Eriksson's Vitamin Mix(Sigma E-1511), 1.0 mg/L thiamine HCl, 1.5 mg/L 2, 4-D, 0.69 g/LL-proline, 30.0 g/L sucrose, 0.5 g/L MES buffer, 0.85 mg/L silvernitrate, 3.0 mg/L Bialaphos, 100 mg/L carbenicillin, and 6.0 g/L agar,pH 5.8. Embryos were subcultured every three weeks until transgenicevents were identified. Somatic embryogenesis was induced bytransferring a small amount of tissue onto regeneration medium (4.3 g/LMS salts (Gibco 11117), 5.0 ml/L MS Vitamins Stock Solution, 100 mg/Lmyo-inositol, 0.1M ABA, 1 mg/L IAA, 0.5 mg/L zeatin, 60.0 g/L sucrose,1.5 mg/L Bialaphos, 100 mg/L carbenicillin, 3.0 g/L Gelrite, pH 5.6) andincubation in the dark for two weeks at 28° C. All material with visibleshoots and roots were transferred onto media containing 4.3 g/L MS salts(Gibco 11117), 5.0 ml/L MS Vitamins Stock Solution, 100 mg/Lmyo-inositol, 40.0 g/L sucrose, 1.5 g/L Gelrite, pH 5.6, and incubatedunder artificial light at 28° C. One week later, plantlets were movedinto glass tubes containing the same medium and grown until they weresampled and/or transplanted into soil.

Example 2 Transient Expression of BBM Enhances Transformation

Parameters of the transformation protocol can be modified to ensure thatthe BBM activity is transient. One such method involves precipitatingthe BBM-containing plasmid in a manner that allows for transcription andexpression, but precludes subsequent release of the DNA, for example, byusing the chemical PEI.

In one example, the BBM plasmid is precipitated onto gold particles withPEI, while the transgenic expression cassette (UBI::moPAT˜GFPm::PinII;moPAT is the maize optimized PAT gene) to be integrated is precipitatedonto gold particles using the standard calcium chloride method.

Briefly, gold particles were coated with PEI as follows. First, the goldparticles were washed. Thirty-five mg of gold particles, 1.0 in averagediameter (A.S.I. #162-0010), were weighed out in a microcentrifuge tube,and 1.2 ml absolute EtOH was added and vortexed for one minute. The tubewas incubated for 15 minutes at room temperature and then centrifuged athigh speed using a microfuge for 15 minutes at 4° C. The supernatant wasdiscarded and a fresh 1.2 ml aliquot of ethanol (EtOH) was added,vortexed for one minute, centrifuged for one minute, and the supernatantagain discarded (this is repeated twice). A fresh 1.2 ml aliquot of EtOHwas added, and this suspension (gold particles in EtOH) was stored at−200° C. for weeks. To coat particles with polyethylimine (PEI; Sigma#P3143), 250 μl of the washed gold particle/EtOH mix was centrifuged andthe EtOH discarded. The particles were washed once in 100 μl ddH2O toremove residual ethanol, 250 μl of 0.25 mM PEI was added, followed by apulse-sonication to suspend the particles and then the tube was plungedinto a dry ice/EtOH bath to flash-freeze the suspension, which was thenlyophilized overnight. At this point, dry, coated particles could bestored at −80° C. for at least 3 weeks. Before use, the particles wererinsed 3 times with 250 μl aliquots of 2.5 mM HEPES buffer, pH 7.1, with1× pulse-sonication, and then a quick vortex before each centrifugation.The particles were then suspended in a final volume of 250 μl HEPESbuffer. A 25 μl aliquot of the particles was added to fresh tubes beforeattaching DNA. To attach uncoated DNA, the particles werepulse-sonicated, then 1 μg of DNA (in 5 μl water) was added, followed bymixing by pipetting up and down a few times with a Pipetteman andincubated for 10 minutes. The particles were spun briefly (i.e. 10seconds), the supernatant removed, and 60 μl EtOH added. The particleswith PEI-precipitated DNA-1 were washed twice in 60 μl of EtOH. Theparticles were centrifuged, the supernatant discarded, and the particleswere resuspended in 45 μl water. To attach the second DNA (DNA-2),precipitation using TFX-50 was used. The 45 μl of particles/DNA-isuspension was briefly sonicated, and then 5 μl of 100 ng/μl of DNA-2and 2.5 μl of TFX-50 were added. The solution was placed on a rotaryshaker for 10 minutes, centrifuged at 10,000 g for 1 minute. Thesupernatant was removed, and the particles resuspended in 60 μl of EtOH.The solution was spotted onto macrocarriers and the gold particles ontowhich DNA-1 and DNA-2 had been sequentially attached were delivered intoscutellar cells of 10 DAP Hi-II immature embryos using a standardprotocol for the PDS-1000. For this experiment, the DNA-1 plasmidcontained a UBI::RFP::pinII expression cassette, and DNA-2 contained aUBI::CFP::pinII expression cassette. Two days after bombardment,transient expression of both the CFP and RFP fluorescent markers wasobserved as numerous red & blue cells on the surface of the immatureembryo. The embryos were then placed on non-selective culture medium andallowed to grow for 3 weeks before scoring for stable colonies. Afterthis 3-week period, 10 multicellular, stably-expressing blue colonieswere observed, in comparison to only one red colony. This demonstratedthat PEI-precipitation could be used to effectively introduce DNA fortransient expression while dramatically reducing integration of thePEI-introduced DNA and thus reducing the recovery of RFP-expressingtransgenic events. In this manner, PEI-precipitation can be used todeliver transient expression of BBM and/or WUS2.

For example, the particles are first coated with UBI::BBM::pinII usingPEI, then coated with UBI::moPAT˜YFP using TFX-50, and then bombardedinto scutellar cells on the surface of immature embryos. PEI-mediatedprecipitation results in a high frequency of transiently expressingcells on the surface of the immature embryo and extremely lowfrequencies of recovery of stable transformants (relative to the TFX-50method). Thus, it is expected that the PEI-precipitated BBM cassetteexpresses transiently and stimulates a burst of embryogenic growth onthe bombarded surface of the tissue (i.e. the scutellar surface), butthis plasmid will not integrate. The PAT˜GFP plasmid released from theCa++/gold particles is expected to integrate and express the selectablemarker at a frequency that results in substantially improved recovery oftransgenic events. As a control treatment, PEI-precipitated particlescontaining a UBI::GUS::pinII (instead of BBM) are mixed with thePAT˜GFP/Ca++ particles. Immature embryos from both treatments are movedonto culture medium containing 3 mg/l bialaphos. After 6-8 weeks, it isexpected that GFP+, bialaphos-resistant calli will be observed in thePEI/BBM treatment at a much higher frequency relative to the controltreatment (PEI/GUS).

As an alternative method, the BBM plasmid is precipitated onto goldparticles with PEI, and then introduced into scutellar cells on thesurface of immature embryos, and subsequent transient expression of theBBM gene elicits a rapid proliferation of embryogenic growth. Duringthis period of induced growth, the explants are treated withAgrobacterium using standard methods for maize (see Example 1), withT-DNA delivery into the cell introducing a transgenic expressioncassette such as UBI::moPAT˜GFPm::pinII. After co-cultivation, explantsare allowed to recover on normal culture medium, and then are moved ontoculture medium containing 3 mg/l bialaphos. After 6-8 weeks, it isexpected that GFP+, bialaphos-resistant calli will be observed in thePEI/BBM treatment at a much higher frequency relative to the controltreatment (PEI/GUS).

It may be desirable to “kick start” callus growth by transientlyexpressing the BBM and/or WUS2 polynucleotide products. This can be doneby delivering BBM and WUS2 5′-capped polyadenylated RNA, expressioncassettes containing BBM and WUS2 DNA, or BBM and/or WUS2 proteins. Allof these molecules can be delivered using a biolistics particle gun. Forexample 5′-capped polyadenylated BBM and/or WUS2 RNA can easily be madein vitro using Ambion's mMessage mMachine kit. RNA is co-delivered alongwith DNA containing a polynucleotide of interest and a marker used forselection/screening such as Ubi::moPAT˜GFPm::PinII. It is expected thatthe cells receiving the RNA will immediately begin dividing more rapidlyand a large portion of these will have integrated the agronomic gene.These events can further be validated as being transgenic clonalcolonies because they will also express the PAT˜GFP fusion protein (andthus will display green fluorescence under appropriate illumination).Plants regenerated from these embryos can then be screened for thepresence of the polynucleotide of interest.

Example 3 DNA Shuffling to Create Variants of LIG3-4 Meganuclease

A. LIG3-4 Meganuclease and LIG3-4 Recognition Sequence

An endogenous maize genomic target site comprising the LIG3-4recognition sequence (SEQ ID NO: 2) was selected for design of a customdouble-strand break inducing agent. The LIG3-4 recognition sequence is a22 bp polynucleotide having the following sequence:ATATACCTCACACGTACGCGTA (SEQ ID NO: 2).

Wild type I-CreI meganuclease (SEQ ID NO: 3) was modified to produce theLIG3-4 meganuclease designed to recognize the LIG3-4 recognitionsequence as described in US patent publication 2009-0133152 A1.Wild-type I-CreI meganuclease is a homodimer. In order to recognize theLIG3-4 recognition sequence, different substitutions were made to eachmonomer and the coding sequences for each monomer were joined by alinker sequence to produce a single-chain fusion polypeptide (LIG3-4,SEQ ID NO: 1)

B. Creation of LIG3-4 Meganuclease Variants

Variants of the LIG3-4 meganuclease were created through gene shufflingmethods. Gene shuffling is an iterative process consisting of discretecycles termed “rounds”. Each round is a cycle of parent selection,library construction, gene evaluation and hit selection. The best hitsfrom one round become the parental genes for the next round.

The first phase of LIG3-4 meganuclease optimization was designed tointroduce amino acid substitutions as found in naturally occurringmeganuclease proteins. Shuffled gene variant libraries were made basedon the LIG3-4 protein template (SEQ ID NO: 1) using techniques includingfamily shuffling, single-gene shuffling, back-crossing, semi-syntheticand synthetic shuffling (Zhang J-H et al. (1997) Proc Natl Acad Sci 94,4504-4509; Crameri et al. (1998) Nature 391: 288-291; Ness et al. (2002)Nat Biotech 20:1251-1255). Libraries were based on phylogenetic sequencediversity, random mutagenesis, and structural features based on thecrystal structure of I-CreI in Protein Data Bank (PDB). Phylogeneticdiversity of several meganuclease proteins (SEQ ID NO: 4-13), includingI-CreI (SEQ ID NO: 3) is shown in FIG. 1A-FIG. 1B. Diversity is definedas the amino acids present within the set of proteins at any positionwhere all proteins do not contain the identical amino acid.

The shuffling process resulted in generation of LIG3-4 variants withrecombinations of amino acid modifications, unintended amino acidmodifications due to mutagenic PCR, deletions, and insertions (SEQ IDNOs:14-38). Corresponding DNA sequences for expression of thesemeganucleases in yeast are shown in SEQ ID NOs: 40-65).

Example 4 Yeast Screening System for Identification of MeganucleaseVariants with Increased Activity

Yeast screening strains were generated as hosts for the identificationof meganuclease variants. The yeast Ade2 gene (Genetika 1987 Jul. 23(7):1141-8) (SEQ ID NO: 82) was used as a visible marker as well as aselection in the scheme depicted in FIG. 2. Gene fragments correspondingto the first 1000 nucleotides of the Ade2 coding sequence (SEQ ID NO:83) (Ade2 5′ fragment) and the last 1011 nucleotides of the Ade2 codingsequence (Ade2 3′ fragment) were disrupted by a fragment including theyeast ura3 gene and meganuclease recognition sites. Three versions ofthe construct depicted in FIG. 2 were used. Plasmid pHD1327 (SEQ ID NO:84) included the ZM6.3, ZM6.22, MHP42, MHP107 and LIG3-4 recognitionsites. pVER8145 included the LIG3-4 recognition site, and pVER8189included the MHP14, MHP77 and LIG3-4 recognition sites. There are 305nucleotides of sequence duplication between the Ade2 5′ fragment and theAde2 3′ fragment. The resulting constructs were used to replace the Ade2gene (chromosome 15 nucleotide position 566193-564480) of yeast strainBY4247. The resulting yeast screening strains VER8145, VER8189 andHD1327 can be characterized as BY4742 MATa his3delta1 leu2delta0lys2delta0 ura3delta0 Gal2+).If meganuclease cutting occurs between theduplicated sequences, homologous recombination can occur, resulting in afunctional Ade2 gene.

The generation of a functional Ade2 gene can be used as a selection:when yeast cells are grown on media lacking adenine, only those with afunctional Ade2 gene are able to grow.

The generation of a functional Ade2 gene can also be used as a screen.Yeast cells with a functional Ade2 gene are white, whereas those lackingAde2 function exhibit red pigmentation due to accumulation of ametabolite earlier in the adenine biosynthetic pathway resulting in redcolonies with white sectors as shown in FIGS. 2 and 3. The degree ofwhite sectoring, sometimes extending to entire colonies, indicates theamount of meganuclease cutting activity. Since the sectoring phenotypeis a qualitative measure of meganuclease activity, a 0-4 numericalscoring system was implemented. As shown in FIG. 3, a score of 0indicates that no white sectors (no meganuclease cutting) were observed;a score of 4 indicates completely white colonies (complete cutting ofthe recognition site); scores of 1-3 indicate intermediate whitesectoring phenotypes (and intermediate degrees of recognition sitecutting).

Example 5 Meganuclease Expression Plasmid

A meganuclease expression plasmid was constructed using the plasmidp415GAL1 (ATCC; Nucleic Acids Res. 1994 Dec. 25; 22(25):5767-8). TheLIG3-4 coding sequence was PCR amplified using primers MN031 (SEQ ID NO:66) and MN022 (SEQ ID NO: 67) and inserted in p415GAL1 as an XbaI-XhoIrestriction fragment. The resulting construct (pVER8134; SEQ ID NO: 68)is shown in FIG. 4. The meganuclease expression plasmid contains acentromeric replication origin and a leu2 nutritional marker for growthin yeast as well as the F1 replication origin and an ampicillinantibiotic resistance gene for growth in E. coli. The meganucleaseexpression cassette consists of the galactose-inducible GAL1 promoterand the CYC1 terminator. The meganuclease coding sequence was precededwith a nuclear localization signal (SEQ ID NO: 69) encoding a 9 aminoacid amino-terminal (MAPKKKRKV, SEQ ID NO: 70) and a carboxy-terminal 6×histidine tag (SEQ ID NO: 71) to aid protein purification.

Similar meganuclease expression plasmids were constructed by exchangingthe LIG3-4 meganuclease (nucleotide positions 500-1549 of pVER8134, SEQID NO: 68) with a variant meganuclease.

Example 6 Transformation of the Yeast Screening Strain (YSS) andScreening for Meganuclease Activity in Yeast

Shuffled meganuclease libraries (comprising the variant meganucleases)were inserted in the expression vector pVER8134 as described in Example5 and transformed into a yeast screening strain comprising thecorresponding meganuclease recognition site (Example 3) by the followingprocedure.

A 3 mL culture of selective media (MP Biomedical) was inoculated with asingle colony of the yeast screening strain and grown at 30° C.overnight. On the following day, a 50 ml YPD culture (MP Biomedical) wasstarted with 2 ml of the overnight culture and grown at 30° C.overnight. On the following day, the cells were harvested bycentrifugation at 4000 rpm. The cells were resuspended in 100 ml icecold water and centrifuged again. The cells were then washed in 1.2Msorbitol, followed by treatment with 2 ml of 10 mM Tris pH 8.0, 1 mMEDTA, 100 mM Lithium acetate, 10 mM DTT, 0.6M sorbitol for 30 minutes at30° C. with shaking. The cells were recovered by centrifugation, washedin 40 ml 1.2M sorbitol and finally resuspended in 250 microliters of1.2M sorbitol. 50 microliter aliquots were transferred to test tubes onice. Up to 5 microliters of DNA (100-500 nanograms) were added. Thesuspension was transferred to a 0.2 cm electroporation cuvette, on ice.Electroporation was performed with a pulse charge at 1.5 kV, 200 ohms,25 microF (pulse time of 5 milliseconds). 1 mL YPD media (MP Biomedical)was added and the cells were allowed to recover at 30° C. for 1-2 hr.The cells were centrifuged, resuspended in 100 uL 1M sorbitol and platedon selective media lacking leucine and containing 2% galactose. Theresulting yeast colonies were incubated at various temperatures rangingfrom 22 to 37 degrees Celsius for 7-10 days. I-CreI and meganucleasesderived from it have maximal activity at or above 37 degrees Celsius.Screening was performed at a range of temperatures from 22 to 37 degreesin order to observe increases in activity at lower temperatures whichare relevant to certain biological systems (eg. plant cells, plant cellcultures, etc). At that time the red/white sectoring phenotype,indicative of meganuclease activity was observed. Colonies withincreased white sectoring over the parental meganuclease (indicatingcolonies expressing a meganuclease with increased meganucleaseactivity), also referred to as “hits” and sometimes completely white,were isolated for further analysis.

These potential “hits” were grown in liquid media to increase the celldensity. DNA was extracted and used to transform E. coli. Plasmid DNAwas extracted from E. coli cultures. The plasmid DNA corresponding tothe potential hits was again transformed into the yeast screening strainas described above.

If the increase in white sectoring phenotype in yeast cells comprisingthe variant meganuclease (when compared to yeast comprising the parentalmeganuclease) was repeated, the variant was declared a “confirmed hit”.Meganuclease coding sequences were determined for confirmed hits. Eachconfirmed hit represents a variant meganuclease and was assigned ameganuclease activity score at various temperatures based on the 0-4scale described in Example 4.

Table 2 shows the activity of LIG3-4 and LIG3-4 variant meganucleases inYeast Screening Strain VER8145 assayed at 22° C. and 30° C. with 2%galactose. A score of 0 indicates that no white sectors (no cuttingindicating no meganuclease activity) was observed; a score of 4indicates completely white colonies (complete cutting of the recognitionsite indicating high meganuclease activity); scores of 1-3 indicateintermediate white sectoring phenotypes (and intermediate degrees ofrecognition site cutting) was indicative of intermediate meganucleaseactivity.

TABLE 2 Activity of LIG3-4 and LIG3-4 variant Meganucleases in YeastScreening Strain assayed at 22° C. and 30° C. Assay score Assay scoreSEQ ID NO: Meganuclease 22° C. 30° C. 1 LIG3-4 0 2 27 LIG3-4(B65) 4 4 28LIG3-4(B70) 4 4 31 LIG3-4(B75) 4 4 32 LIG3-4(B76) 4 4 30 LIG3-4(B73) 4 434 LIG3-4(B82) 4 4 33 LIG3-4(B78) 4 4 18 LIG3-4(B1) 3.5 4 15 LIG3-4(15)3 4 38 LIG3-4(D8) 2.5 4 19 LIG3-4(B15) 2.5 4 35 LIG3-4(C1) 2 4 29LIG3-4(B71) 2 4 24 LIG3-4(B39) 1 4 20 LIG3-4(B16) 0.5 4 37 LIG3-4(D7)0.5 4 23 LIG3-4(B38) 0 4 25 LIG3-4(B40) 0 4 22 LIG3-4(B36) 0 4 21LIG3-4(B24) 0 4 26 LIG3-4(B55) 0 4 16 LIG3-4(A4) 0 3.5 36 LIG3-4(D5) 1 314 LIG3-4(7) 1 3 17 LIG3-4(A6) 0 3

Alignment of the LIG3-4 variants relative to the LIG3-4 parent(LIG3-4.pro) is shown in FIG. 5A-FIG. 5E.

The various assay conditions are indicative of meganuclease activity,allowing a precise ranking of the shuffled variants by activity. Largeincreases in meganuclease activity (high scores) were observed. Completecutting of the recognition site was observed with some variants even atthe low temperature of 22° C. This is significant because the optimaltemperature for I-Cre type meganucleases is 37° C., whereas the optimaltemperature for certain biological systems (eg. plant cell cultures) isin the range of 22-25 degrees Celsius. Hence, these variantmeganucleases that can cut at lower temperatures are better suited tofunction well in plant systems when compared to the parental I-Cre typemeganuclease.

Table 3A and 3B represent the amino acid modifications of LIG3-4variants relative to the LIG3-4 parental meganuclease.

TABLE 3A Amino acid modifications of LIG3-4 variants relative to theLIG3-4. SEQ ID NO Meganuclease 16 19 22 50 54 56 59 71 81 103 121 132153 1 LIG3-4 F G S Q F D V G I N K I D 27 LIG3-4(B65) _(—) _(—) C K _(—)_(—) _(—) K _(—) _(—) _(—) _(—) _(—) 28 LIG3-4(B70) _(—) _(—) C K _(—)_(—) _(—) K _(—) _(—) _(—) _(—) _(—) 31 LIG3-4(B75) _(—) _(—) C K _(—)_(—) _(—) P _(—) _(—) _(—) _(—) _(—) 32 LIG3-4(B76) _(—) _(—) C K _(—)_(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) 30 LIG3-4(B73) _(—) _(—) C _(—)_(—) _(—) _(—) K _(—) _(—) _(—) _(—) _(—) 34 LIG3-4(B82) _(—) _(—) C K_(—) _(—) _(—) K _(—) _(—) _(—) _(—) _(—) 33 LIG3-4(B78) _(—) _(—) C_(—) _(—) _(—) _(—) P _(—) _(—) _(—) _(—) _(—) 18 LIG3-4(B1) _(—) _(—) C_(—) _(—) _(—) _(—) P _(—) _(—) _(—) _(—) _(—) 15 LIG3-4(15) _(—) S _(—)_(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) 38 LIG3-4(D8) _(—)_(—) _(—) _(—) I _(—) H _(—) _(—) _(—) G _(—) _(—) 19 LIG3-4(B15) _(—)_(—) C _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) 35 LIG3-4(C1)_(—) _(—) C _(—) _(—) _(—) _(—) K _(—) _(—) _(—) _(—) _(—) 29LIG3-4(B71) _(—) _(—) C _(—) _(—) _(—) _(—) K _(—) _(—) _(—) _(—) _(—)24 LIG3-4(B39) _(—) _(—) C _(—) _(—) _(—) _(—) P _(—) _(—) _(—) _(—)_(—) 20 LIG3-4(B16) _(—) _(—) C K _(—) _(—) _(—) _(—) _(—) _(—) _(—)_(—) _(—) 37 LIG3-4(D7) _(—) _(—) _(—) R _(—) L _(—) _(—) K _(—) _(—)_(—) L 23 LIG3-4(B38) _(—) _(—) C _(—) _(—) _(—) _(—) _(—) _(—) _(—)_(—) _(—) _(—) 25 LIG3-4(B40) _(—) _(—) _(—) K _(—) _(—) _(—) _(—) _(—)_(—) _(—) _(—) _(—) 22 LIG3-4(B36) _(—) _(—) _(—) _(—) _(—) _(—) _(—) K_(—) _(—) _(—) _(—) _(—) 21 LIG3-4(B24) _(—) _(—) C _(—) _(—) _(—) _(—)_(—) _(—) _(—) _(—) _(—) 26 LIG3-4(B55) _(—) _(—) C _(—) _(—) _(—) _(—)_(—) _(—) _(—) _(—) _(—) _(—) 16 LIG3-4(A4) _(—) _(—) _(—) _(—) _(—)_(—) _(—) _(—) _(—) _(—) _(—) V _(—) 36 LIG3-4(D5) _(—) _(—) _(—) _(—)_(—) _(—) _(—) _(—) K V _(—) _(—) M 14 LIG3-4(7) I _(—) _(—) _(—) _(—)_(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) 17 LIG3-4(A6) _(—) _(—) _(—)_(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) V _(—)

TABLE 3B (continued from Table 3A) SEQ ID NO Meganuclease 185 209 222244 246 258 281 308 316 319 345 346 1 LIG3-4 A S F K V G F K V I K K 27LIG3-4(B65) _(—) _(—) _(—) _(—) _(—) K _(—) _(—) _(—) _(—) _(—) _(—) 28LIG3-4(B70) _(—) _(—) _(—) _(—) _(—) P _(—) _(—) _(—) _(—) _(—) _(—) 31LIG3-4(B75) _(—) _(—) _(—) _(—) _(—) K _(—) _(—) _(—) _(—) _(—) _(—) 32LIG3-4(B76) _(—) _(—) _(—) _(—) _(—) K _(—) _(—) _(—) _(—) _(—) _(—) 30LIG3-4(B73) _(—) _(—) _(—) _(—) _(—) K _(—) _(—) _(—) _(—) _(—) _(—) 34LIG3-4(B82) _(—) C _(—) _(—) _(—) K _(—) _(—) _(—) _(—) _(—) _(—) 33LIG3-4(B78) _(—) _(—) _(—) _(—) _(—) K _(—) _(—) _(—) _(—) N N 18LIG3-4(B1) _(—) _(—) _(—) _(—) _(—) K _(—) _(—) _(—) _(—) _(—) _(—) 15LIG3-4(15) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—)38 LIG3-4(D8) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—)_(—) 19 LIG3-4(B15) _(—) _(—) _(—) _(—) _(—) K _(—) _(—) _(—) _(—) _(—)_(—) 35 LIG3-4(C1) _(—) _(—) L _(—) _(—) _(—) _(—) _(—) _(—) _(—) N N 29LIG3-4(B71) _(—) _(—) _(—) _(—) _(—) P _(—) _(—) _(—) _(—) _(—) _(—) 24LIG3-4(B39) _(—) _(—) _(—) _(—) _(—) P _(—) _(—) _(—) _(—) N N 20LIG3-4(B16) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—)37 LIG3-4(D7) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—)_(—) 23 LIG3-4(B38) _(—) _(—) _(—) _(—) _(—) P _(—) _(—) _(—) _(—) _(—)_(—) 25 LIG3-4(B40) _(—) _(—) _(—) _(—) _(—) K _(—) _(—) _(—) _(—) _(—)_(—) 22 LIG3-4(B36) _(—) _(—) _(—) _(—) _(—) K _(—) _(—) _(—) _(—) _(—)_(—) 21 LIG3-4(B24) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—)_(—) _(—) 26 LIG3-4(B55) _(—) C _(—) _(—) _(—) K _(—) _(—) _(—) _(—)_(—) _(—) 16 LIG3-4(A4) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) _(—) V_(—) _(—) 36 LIG3-4(D5) _(—) _(—) _(—) _(—) H _(—) _(—) G _(—) _(—) _(—)_(—) 14 LIG3-4(7) G _(—) _(—) E _(—) _(—) _(—) _(—) A _(—) _(—) _(—) 17LIG3-4(A6) _(—) _(—) _(—) _(—) _(—) _(—) Y _(—) _(—) _(—) _(—) _(—)

Example 7 Meganuclease Protein Production in E. coli

In order to further confirm and quantify the activity of meganucleasevariants, meganuclease protein was produced in E. coli and subjected toin vitro cutting assay on plasmid or corn genomic DNA containing themeganuclease recognition site. Total DNA was extracted from yeaststrains harboring the meganuclease variants. The meganuclease codingsequence was PCR amplified and inserted in the expression vector pQE80(QIAgen). The resulting plasmid was transformed into E. coli strain BL21(Stratagene) with growth on LB media containing 100 ppm carbenicillin. Asuspension of cells was prepared from the solid media and used toinoculate a 50 ml culture of 2×YT media containing 100 ppm carbenicillinat an optical density of 0.2. The cultures were grown at 37 degrees.When the optical density reached 0.8, protein expression was induced byaddition of IPTG. The temperature was adjusted to 20 degrees, and theculture was grown for an additional 2 hours. E. coli cells wereharvested by centrifugation, resuspended in Buffer 1 (50 mM Tris pH8,500 mM NaCl, 10 mM imidizole) and lysed by sonication. Cell debris wasremoved by centrifugation. The supernatant was transferred to adisposable column loaded with 0.5 ml Nickel-NTA Superflow resin(QIAgen). The column was washed with 4 ml Buffer 2 (50 mM Tris pH8, 500mM NaCl, 60 mM imidizole). Purified meganuclease protein was eluted with0.6 ml Buffer 4 (50 mM Tris pH8, 500 mM NaCl, 400 mM imidizole). Themeganuclease protein was transferred to a Vivaspin500 concentrator.Buffer exchange and concentration with SAB buffer (25 mM Tris pH8, 100mM NaCl, 10 mM MgCl₂, 5 mM EDTA) containing 50% glycerol, 0.5 mMdithiothreitol was performed. A final volume of approximately 0.1 ml ofpurified meganuclease protein solution was recovered. Bovine serumalbumin was added to a final concentration of 100 microgram permilliliter.

Example 8 In Vitro Assay for Meganuclease Activity

Meganuclease protein was isolated as described in Example 7. Proteinconcentration was determined visually on Nu-PAGE gels (LifeTechnologies) by calculating and then comparing band intensity withserially diluted samples of known concentration. DNA concentration wasdetermined using a Hoechst dye fluorometric assay. Time-coursedigestions were carried out on plasmid DNA containing the meganucleaserecognition site at 37° C., 28° C., and 23° C. with 25 nM of purifiedmeganuclease protein and 0.25 nM of linearized plasmid substrate indigestion buffer (100 mM Tris-HCl (pH 7.9)/100 mM NaCl/10 mM MgCl₂/1 mMDTT/5 mM EDTA) in a final volume of 80 ul. 20 μl time-points were takenat 0, 25, 50, and 75 minutes and stopped with an equal volume of stopbuffer (100 mM Tris-HCl, pH 8.0/600 mM NaCl/2% SDS/100 mM EDTA/1 mg ofproteinase K per ml), incubated at 50° C. for 30-45 minutes, andpurified with a Qiagen PCR purification column per the manufacturer'sinstruction. To quantify the % digestion of each sample or loss ofmeganuclease recognition sites, real-time PCR was performed on 1 μl ofpurified plasmid DNA diluted 50-fold in water with a TaqMan assayspanning the meganuclease recognition site. The loss of meganucleaserecognition sites was calculated via the ΔΔCt method relative to aninternal control TaqMan assay. The 0 minute timepoint or mock controlwas used as the calibrator. Timed digestions were carried out on genomicDNA at 37° C., 28° C., and 23° C. with 6.07 ug of corn genomic DNA and16 nM of purified meganuclease protein in a final volume of 80 ul. After50 minutes, digestion reactions were stopped as described above andpurified by phenol/chloroform extraction and ethanol precipitated in thepresence of 0.2M NaCl. Precipitated genomic DNA was washed twice with70% ethanol, dried, and resuspended in 34 μl of water. The percentdigestion of each sample was quantified by real-time PCR as describedabove for plasmid substrate except 1 μl of undiluted genomic DNA wasassayed by real-time PCR. Since the cleavage activity of the I-CreIendonuclease has been demonstrated to be sensitive to temperatures below37° C. (Wang, J., Kim, H., Yuan, X. and Herrin, D. (1997) Nucleic AcidsRes. 25, 3767-3776.), in vitro assays to assess cleavage activity of theI-CreI derived parental meganuclease and its variants were carried outat 37° C., 28° C., and 23° C.

In-Vitro Meganuclease Activity of LIG3-4 and LIG3-4 Variants

On plasmid DNA containing the LIG3-4 recognition site, LIG3-4(B65) wasthe most active variant sustaining cleavage activity at 23° C. whilelittle if any cleavage was detected for LIG3-4 and only slight cleavagewas detected for LIG3-4(7) and LIG3-4(15) (FIG. 6A). At 28° C., hit15and hit 7 achieved 66% and 50% cleavage, respectively, after 75 minuteswhile only slight cleavage was detected for LIG3-4 (FIG. 6B). At 37° C.,hit7 demonstrated cleavage activity similar to LIG3-4 while hit15 andB65 cleaved the plasmid substrate more rapidly and to a greater extent(FIG. 6C). Based on plasmid DNA cleavage, all of the shuffled variantswere more active than LIG3-4 with B65 being the most active variantfollowed in activity by hit15 and then hit7. These data closely mimickedthe observations in the yeast assay.

The activity ranking established on plasmid DNA was conserved whengenomic DNA cleavage was monitored. At 23° C., B65 maintainedsignificant activity, cleaving 69% of its genomic substrate (FIG. 7A).At 28° C., no cleavage activity was detected for LIG3-4 while B65, hit15and hit7 obtained 94%, 33% and 24% cleavage, respectively (FIG. 7B). At37° C., LIG3-4 exhibited 47% cleavage at the LIG3-4 genomic recognitionsite while B65, hit15 and hit7 achieved 99%, 92% and 76% cleavage,respectively (FIG. 7C). Again, the data from in vitro cutting of maizegenomic DNA were consistent with observations in the yeast assay.

Example 9 Analysis of Meganuclease Activity of LIG3-4 Variants in Maize

LIG3-4 variants were created as described in Example 3 and introduced inmaize by particle gun transformation and Agrobacterium-mediatedtransformation.

Three LIG3-4 variants, LIG3-4 (B65) (SEQ ID NO: 27), LIG3-4(15) (SEQ IDNOs: 15) and LIG3-4(7) (SEQ ID NO: 14) showed an increased meganucleaseactivity in yeast (Example 3) and an increased activity in the in vitroassay (Example 8, FIGS. 6A-6C; FIGS. 7A-7C) and were further testedin-vivo for their activity in maize.

A. Vector Construction for Plant Expression Vectors of the MeganucleaseGenes and Repair (Donor) DNAs for Transgene Integration by HomologousRecombination

Genes encoding the meganucleases were codon optimized for expression inmaize using standard molecular biology techniques. The resultingplant-optimized nucleotide sequences were also supplemented with DNAsequences encoding a SV40 nuclear localization signal (SEQ ID NO: 72)and further modified by addition of the potato ST-LS 1 intron to thecoding sequence of the first monomer in order to eliminate itsexpression in E. coli and Agrobacterium. The resulting LIG3-4 variants,LIG3-4(7) (SEQ ID NO: 73), LIG3-4(15) (SEQ ID NO: 74) and LIG3-4 (B65)(SEQ ID NO: 75) were further tested for their activity in maize (invivo).

Vectors comprising expression cassettes for the appropriate meganucleasewere constructed using standard molecular biological techniques. Foreach of the meganucleases, a plant expression vector comprising apolynucleotide encoding one of the meganuclease genes was operablylinked to a maize constitutive promoter.

To achieve site-specific DNA insertions, a repair DNA (donor DNA)containing the gene of interest has to be simultaneously present in thecell in addition to the recognition site and the meganuclease. A vectorPHP46961 (SEQ ID NO: 76) containing a polynucleotide encoding theengineered meganuclease variant LIG3-4(15), and a donor DNA wereconstructed using standard molecular biology techniques. Similar vectorsPHP46949 or PHP47257 were constructed containing the LIG3-4B(65) orLIG3-4(17), meganuclease respectively. The donor DNA contained anherbicide resistance gene (MoPAT, encoding a phosphinothricinacetyltransferase), used as the selection marker for transformation, andwas flanked by two homologous recombination fragments, LIG3-4HR1 (SEQ IDNO: 77) and LIG3-4HR2 (SEQ ID NO: 78), which were about 1 kb longgenomic DNA sequences flanking the meganuclease recognition sites. Avector containing LIG3-4 (PHP43914, produced as described for PHP46961)was also included as control.

The LIG3-4 variants' expression cassettes were also co-integrated intoLBA4404 for Agrobacteria delivery. Vector names were PHP47331 forLIG3-4(B65), PHP47332 for LIG3-4 (15) and PHP47517 for LIG3-4(7),respectively.

Maize immature embryos 9-12 DAP (days after pollination, approximately1.5-2.0 mm in size) from a maize transformable line were used for genetransformation by bombardment (Example 1 and Example 2). The immatureembryos were placed on 560Y medium for 4 hours at 26° C. oralternatively, immature embryos were incubated at temperatures rangingfrom 26° C. to 37° C. for 8 to 24 hours prior to placing on 560Ypreceding bombardment. Developmental genes ODP2 (AP2 domaintranscription factor ODP2 (Ovule development protein 2); US20090328252A1) and Wushel were included in the experiments through co-bombardment(Example 2). Maize immature embryos were transformed with the vectorsPHP43914, PHP46949, PHP46961, and PHP47257.

Maize immature embryos 9-12 DAP (days after pollination, approximately1.5-2.0 mm in size) from a maize transformable line were used for genetransformation by Agrobacterium. No developmental genes ODP2 or Wushelwere included in the infection. Maize immature embryos were transformedwith vectors of PHP47731, PHP47732, and PHP47517.

B. Meganuclease Activity of LIG3-4 Variants in Maize

To examine whether the LIG3-4 variants showed increased meganucleaseactivity in maize when compared to LIG3-4 about 2000 maize immatureembryos were bombarded with plasmid DNA comprising each variant orcontrol. Following bombardment, embryos were incubated on 560P(maintenance medium) at 28° C., then selected on bialophos herbicide.Successful delivery of LIG3-4 and the LIG3-4 variant donor vectors(PHP43914, PHP46949, PHP46961, and PHP47257) conferred bialaphosherbicide resistance, and was used to identify putative events by callusselection on herbicide containing media. Callus tissues and/or plantsregenerated from stable transformants were screened for modification ofthe endogenous LIG3-4 recognition site.

Herbicide-resistant events were screened for modification at themeganuclease target site (comprising the recognition site) by measuringthe target site copy-number using Real time PCR (qPCR). Two copies ofthe target site indicate that both alleles are wild type and that nomodification occurred at the recognition site. If only one copy of thetarget site is detected by qPCR, this means that one allele of thetarget site has changed during repair of the double strand breakgenerated by the LIG3-4 or its shuffle variants, while absence of thetarget site (null or 0) is the result of both alleles bring modified.The copy number can also be in between 1 and 2 due to chimeric nature ofcallus samples. The probe sequence for qPCR of LIG3-4 target site wasATACCTCACACGTACGCG (SEQ ID NO: 79), the LIG3-4_forward primer wasGATTTACGCACCTGCTGGGA (SEQ ID NO: 80) and LIG3-4_reverse primer wasCTGAGCTGTATTCCCGCGCA (SEQ ID NO: 81) The amplicon was approximately 100bp.

Transgenic events with a target site copy number of 0, 1, or between 1and 2 were further analyzed for increased meganuclease activity. Themeganuclease activity was determined by measuring the Target Site (TS)mutation rate. Target site mutation rate was defined as: (number ofevents with target site modification/total number events)*100%. The TSmutation rate for the LIG3-4 meganuclease was 6% (Table 4). The EventRecovery Rate (Table 4) is calculated using number of events recovereddivided by total number of embryos bombarded, and may indicate if ameganuclease has some toxic effect or not. Table 4 shows the effect ofdifferent LIG3-4 variants after bombardment and 6-8 weeks antibioticselection. The meganuclease variants LIG3-4 (7) and LIG3-4 (15) bothyielded significantly higher mutation frequencies when compared to theparental LIG3-4 meganuclease, consistent with observations in the yeastassay and in vitro DNA cutting assays. LIG3-4(B65) also yielded highermutation frequency than the parental LIG3-4, but not as high as theother LIG3-4 variants. This may be due to toxicity associated with thisvery active meganuclease as indicated by the event recovery rate ofLIG3-4 (65).

TABLE 4 Activity of LIG3-4 and LIG3-4 variant meganucleases asdetermined by target site mutation rate (TS mutation rate) in planttissue originated through gene bombardment transformation. MeganucleaseEvent Recovery Rate TS Mutation Rate Insertion LIG3-4 17% 6% Yes LIG3-4(7) 13% 29% Yes LIG3-4 (15) 15% 54% Yes LIG3-4 (B65) 3% 21% Yes

TABLE 5 Activity of LIG3-4 and LIG3-4 variant meganucleases asdetermined by target site mutation rate (TS mutation rate) in planttissue originated through Agrobacterium transformation. MeganucleaseEvent Recovery Rate Mutation Rate Insertion LIG3-4 ~20% 1-3%  No LIG3-4(7) 19% 15% Yes LIG3-4 (15) 11% 34% Yes LIG3-4 (B65) 9% 74% Yes

Table 5 indicates that all three variant meganucleases (LIG3-4 (7),LIG3-4(15) and LIG3-4(B65) showed an increased meganuclease activity (TSmutation rate of 15%, 34% and 74%, respectively) when compared to thecontrol non variant LIG3-4 (TS mutation rate 1 to 3%). The highestincrease in meganuclease activity was observed when plant tissue wasgenerated through Agrobacterium transformation. (Table 5). These dataare very consistent with data obtained in the yeast and in vitro cuttingassays with these variants.

When the meganuclease and gene delivery constructs were introduced viaAgrobacterium-mediated transformation, there was a much smallerreduction in the recovery of transgenic events (higher event recoveryrate in Table 5 when compared to Table 4). This may be due to the factthat less DNA is delivered to the nuclei of the plant cells by thismethod.

Maize calli were also screened for integration of the transgene cassettefrom the donor DNA (PHP43914, PHP46949, PHP46961, PHP47257; agro ofPHP47331, PHP47332, and PHP47517) at the LIG3-4 recognition site throughjunction PCR and selected callus events were regenerated into T0 plants(FIG. 8A-FIG. 8B). When integration occurred, e.g. the donor sequencewas integrated at the recognition site, Insertion is designated as“Yes”. When no integration occurred, Insertion is designated as “no”(Table 4 and 5). Targeting of transgenes to the LIG3-4 locus wasobserved with each LIG3-4 variant delivered by particle bombardment(Table 4. When introduced via Agrobacterium-mediated transformation,each LIG34 variant enabled transgene integration at the target site,whereas the parental LIG34 did not (Insertion YES for variants, NO forLIG3-4; Table 5).

Example 10 Creation of MHP77 and MHP77.3 Variant Meganucleases

A. MHP77 & MHP77.3 Meganucleases and MHP 77 Recognition Site

An endogenous maize genomic target site comprising the MHP77 recognitionsite (SEQ ID NO: 85) was selected for design of a custom double-strandbreak inducing agent. The MHP77 recognition site is a 22 bppolynucleotide located on chromosome 1 and having the followingsequence:

(SEQ ID NO: 85) GGGCGGTATGTATGTCATACTA

Wild type I-CreI meganuclease (SEQ ID NO: 3) was modified to produce twoengineered meganucleases, MHP77 (SEQ ID NO: 86) and MHP77.3 (SEQ ID NO:250), designed to recognize the MHP77 recognition sequence. The designof custom made meganucleases has been described in United States PatentApplication Publication No. US 2007/0117128 A1.

B. MHP77 and MHP77.3 Variant Meganucleases

Variants of the MHP77 meganuclease were created through gene shufflingmethods in a manner similar to how the LIG3-4 variants were created anddescribed in Example 3. This involved the introduction of amino acidmodifications as found in naturally occurring meganuclease proteins andpreviously identified in LIG3-4 variants as well as random mutations.The shuffling process resulted in generation of MHP77 variants withrecombination of amino acid modifications, unintended amino acidmodifications due to mutagenic PCR, deletions, and insertions (SEQ IDNOs: 86-167)

Three variants of the MHP77.3 meganuclease were created by incorporatingthe same amino acid modifications (mutations) of MHP77(L9-02),MHP77(L9-11), or MHP77(L9-12), thus creating MHP77.3 (L9-02) (SEQ ID NO:251), MHP77.3 (L9-11) (SEQ ID NO: 252), and MHP77.3(L9-12) (SEQ ID NO:253). MHP77.3 (15) (SEQ ID NO: 262) contained the exact samenucleotide/amino acid modifications as described for LIG3-4 (15). Theamino acid modifications were introduced into MHP77.3 through standardmolecular biology techniques.

C. MHP77 Variant Meganucleases Activity in Yeast

A total of 79 MHP77 variants with increased activity were confirmed inthe yeast system (as described in Example 6). Increased activity wasobserved across a range of temperatures: 24° C., 28° C., 30° C. and 3737° C., as shown in Table 6. A score of 0 indicates that no whitesectors (no cutting indicating no meganuclease activity) were observed;a score of 4 indicates completely white colonies (complete cutting ofthe recognition site indicating high meganuclease activity); scores of1-3 indicate intermediate white sectoring phenotypes (and intermediatedegrees of recognition site cutting) was indicative of intermediatemeganuclease activity.

TABLE 6 Activity of MHP77 variant Meganucleases in Yeast ScreeningStrain assayed at different temperatures. #Variant 24° C. 28° C. 30° C.37° C. MHP77 0 0 0 0 MHP77 (L15-31) X 4 4 4 MHP77 (L16-11) 4 4 4 4 MHP77(L16-09) 2.5 4 4 4 MHP77 (L16-04) 2 4 4 4 MHP77 (L16-19) 2 4 4 4 MHP77(L16-17) 3 3.5 4 4 MHP77 (L16-23) 1 3.5 4 4 MHP77 (L15-34) 1 3.5 4 4MHP77 (L15-40) 0.5 3.5 4 4 MHP77 (L15-39) 0.5 3.5 4 4 MHP77 (L15-45) 0.53 4 4 MHP77 (L15-29) 0.5 2.5 4 4 MHP77 (L15-06) 0 2 4 4 MHP77 (L16-08) 13 3.5 4 MHP77 (L16-05) 1 3 3.5 4 MHP77 (L16-02) 0.5 2.5 3.5 4 MHP77(L16-24) 0 2.5 3.5 4 MHP77 (L16-21) 0 2.5 3.5 4 MHP77 (L16-14) 0 2.5 3.54 MHP77 (L16-18) 0.5 2 3.5 4 MHP77 (L15-27) 0 2 3.5 4 MHP77 (L9-02) 0 23 4 MHP77 (L16-12) 0 2 3 4 MHP77 (L16-01) 0 2 3 4 MHP77 (L15-05) 0 2 3 4MHP77 (L15-24) 0 2 3 4 MHP77 (L16-06) 0 1 3 4 MHP77 (L16-15) 0 1 3 4MHP77 (L15-33) 0 1 3 4 MHP77 (L16-03) 0 2 2.5 4 MHP77 (L15-47) 0 0 2.5 4MHP77 (L15-46) 0 0 2.5 4 MHP77 (L9-12) 0 1 2 4 MHP77 (L16-16) 0 1 2 4MHP77 (L15-10) 0 1 2 4 MHP77 (L9-03) 0 0.5 2 4 MHP77 (L15-20) 0 0.5 2 4MHP77 (L15-28) 0 0 2 4 MHP77 (L15-21) 0 0 2 4 MHP77 (L15-13) 0 0 2 4MHP77 (L9-04) 0 0 1 4 MHP77 (L15-18) 0 0 1 4 MHP77 (L18-01) X 0 0 4MHP77 (L17-12) X 0 0 4 MHP77 (L17-01) X 0 0 4 MHP77 (L15-03) 0 0.5 2 3.5MHP77 (L15-11) 0 0.5 1 3.5 MHP77 (L18-12) X 0 1 3.5 MHP77 (L15-15) 0 0 13.5 MHP77 (L15-12) 0 0 1 3.5 MHP77 (L9-1)* 0 1 2 3 MHP77 (L9-9) 0 0 1 3MHP77 (L9-11) 0 0 1 3 MHP77 (L9-10) 0 0 1 3 MHP77 (L15-02) 0 0 1 3 MHP77(L15-08) 0 0 0.5 3 MHP77 (L16-07) 0 0 0 3 MHP77 (L15-35) 0 0 0 2.5 MHP77(L13-12) 0 0 0 2.5 MHP77 (L113-01) 0 0 0 2.5 MHP77 (L9-06) 0 0.5 0.5 2MHP77 (L15-42) 0 0 0 2 MHP77 (L15-41) 0 0 0 2 MHP77 (L15-36) 0 0 0 2MHP77 (L15-30) 0 0 0 2 MHP77 (L112-03a) 0 0 0 2 MHP77 (L73-02a) 0 0 01.5 MHP77 (L13-10B1) 0 0 0 1.5 MHP77 (L72-08a) X 0 0 1 MHP77 (L72-09a) 00 0 1 MHP77 (L72-01a) 0 0 0 1 MHP77 (L13-08a) 0 0 0 1 MHP77 (L13-06) 0 00 1 MHP77 (L13-02) 0 0 0 1 MHP77 (L13-01a) 0 0 0 1 MHP77 (L73-05a) 0 0 00.5 MHP77 (L15-43) 0 0 0 0.5 MHP77 (L13-04) 0 0 0 0.5 MHP77 (L13-11) 0 00 0.5 MHP77 (L15-23) 0 0 0 0 MHP77 (L15-16) 0 0 0 0

Large increases in meganuclease activity (high scores) were observed.Complete cutting of the recognition site was observed with some variantseven at the low temperature of 22° C. (see MHP77(L16-11) Table 6).

FIG. 9A-FIG. 9N show the amino acid modifications of MHP77 variantsrelative to the MHP77 parental meganuclease. A (-) indicates that theamino acid is identical to the MHP77 reference sequence.

Example 11 Analysis of MHP77 and MHP77.3 Meganuclease Variants in Maize

Genes encoding the MHP77 and MHP77.3 engineered meganucleases (Example10) were optimized for expression in plants. The engineered meganucleaseexpression cassettes contained the maize codon-optimized nucleotidesequences for better performance in maize cells. The meganuclease genesequences were also supplemented with DNA sequences encoding a SV40nuclear localization signal resulting in the plant optimized sequence ofSEQ ID NO: 254 for MHP77 and SEQ ID NO: 255 for MHP77.3. The maizeubiquitin promoter and the potato proteinase inhibitor II geneterminator sequences completed the endonuclease gene designs.

The plant optimized nucleotide sequence for the MHP77 and MHP77.3variants were MHP77(L9-02) (SEQ ID NO: 256), MHP77(L9-11) (SEQ ID NO:257), MHP77(L9-12) (SEQ ID NO: 258), MHP77.3 (L9-02) (SEQ ID NO: 259,MHP77.3 (L9-11) (SEQ ID NO: 260), and MHP77.3(L9-12) (SEQ ID NO: 261)and MHP77(15) (SEQ ID NO: 263).

A. Vector Construction for Plant Expression Vectors of the MeganucleaseGenes and Repair (Donor) DNAs for Transgene Integration by HomologousRecombination

Vectors comprising expression cassettes for the appropriate meganucleasewere constructed using standard molecular biological techniques. Foreach of the meganucleases, a plant expression vector comprising apolynucleotide encoding one of the meganuclease genes was operablylinked to a maize constitutive promoter.

To achieve site-specific DNA insertions, a repair DNA (donor DNA)containing the gene of interest has to be simultaneously present in thecell in addition to the recognition site and the meganuclease. Vectorssimilar to PHP46961 (SEQ ID NO: 76) described in Example 9, butcontaining a polynucleotide encoding the meganuclease variantMHP77(L9-11), MHP77(L9-12), MHP77(L9-02), MHP77.3(L9-11),MHP77.3(L9-12), MHP77.3(L9-02), or MHP77.3(15); and a donor DNA wereconstructed using standard molecular biology techniques. These vectorswere referred to as PHP53132, PHP53134, PHP53136, PHP53133, PHP53135,PHP53137 and PHP50239. The donor DNA contained an herbicide resistancegene used as the selection marker for transformation. The herbicideresistance gene MoPAT encodes a phosphinothricin acetyltransferase, andwas flanked by two homologous recombination fragments, MHP77HR1 (SEQ IDNO: 264) and MHP77HR2 (SEQ ID NO: 265), which were about 1 kb longgenomic DNA sequences flanking the meganuclease recognition sites.

Maize immature embryos 9-12 DAP (days after pollination, approximately1.5-2.0 mm in size) from a maize transformable line were used for genetransformation by bombardment (Example 1 and Example 2). The immatureembryos were placed on 560Y medium for 4 hours at 26° C. oralternatively, immature embryos were incubated at temperatures rangingfrom 26° C. to 37° C. for 8 to 24 hours prior to placing on 560Ypreceding bombardment. Developmental genes ODP2 (AP2 domaintranscription factor ODP2 (Ovule development protein 2); US20090328252A1) and Wushel were included in the experiments through co-bombardment(Example 2). Maize immature embryos were transformed with the vectorsPHP53132, PHP53134, PHP53136, PHP53133, PHP53135, PHP53137, andPHP50239.

B. Meganuclease Activity of MHP77 and MHP77.3 Variants in Maize

To examine whether the MHP77 and MHP77.3 meganuclease variants increasedmeganuclease activity when compared to MHP77 or MHP77.3, about 2000maize immature embryos were bombarded with plasmid DNA of each variantand control. Following bombardment, embryos were incubated on 560P(maintenance medium) at 28° C., then selected on Herbicide (bialophos).Successful delivery of the MHP77, MHP77.3 variants donor vectors(PHP45970, PHP50238, PHP53132, PHP53134, PHP53136, PHP53133, PHP53135,PHP53137, or PHP50239) conferred bialaphos herbicide resistance, and wasused to identify putative events by callus selection on herbicidecontaining media. Callus tissues and/or plants regenerated from stabletransformants using standard culture and regeneration conditions werescreened for modification of the endogenous MHP77 recognition site.

Herbicide-resistant events were screened for modification at the targetsite (comprising the MHP77 recognition site) by measuring target sitecopy-number using qPCR as described in Example 9. The probe sequence forqPCR of MHP77 target site was ACTAATTCAAGTGATGGACAAA (SEQ ID NO: 266),the MHP77_forward primer was TCCTTAGGGCGGTATGTATGTCA (SEQ ID NO: 267)and MHP77_reverse primer was CATCGGTCAAAAAACACATAAACTTT (SEQ ID NO:268). The amplicon was approximately 100 bp.

Target site mutation rate (TS mutation rate, Table 7) indirectlymeasures the meganuclease activity. Table 7 shows the effect ofdifferent shuffle variants of MHP77 and shuffle meganuclease afterbombardment and 6-8 weeks antibiotic selection. Table 7 indicates thatall the three shuffled variants of MHP77 meganucleases are more activewhen compared to MHP77 meganuclease. Increased activity of shuffledMHP77 meganuclease also resulted in a reduction of the event recoverywhen compared to the MHP77 (control).

TABLE 7 Activity of MHP77 and MHP77 variant meganucleases as determinedby target site mutation rate (TS mutation rate) in plant tissueoriginated through gene bombardment transformation. Meganuclease EventRecovery Rate TS Mutation Rate Insertion MHP77 (control) 21% 1% noMHP77L9-11 11% 4% no MHP77L9-12 9% 17% yes MHP77L9-02 3% 6% no

TABLE 8 Activity of MHP77.3 and MHP77.3 variant meganucleases asdetermined by target site mutation rate (TS mutation rate) in planttissue originated through Agrobacterium transformation. MeganucleaseTransformation rate Mutation Rate Insertion MHP77.3 14% 11% noMHP77.3(15) 13% 22% yes MHP77.3L9-11 9% 35% yes MHP77.3L9-12 3% 19% yesMHP77.3L9-02 2% 5% no

Table 8 indicates that all the four shuffled variants of MHP77.3meganucleases are more active when compared to the non variant MHP77.3meganuclease. Increased activity of some but not all shuffled MHP77meganuclease resulted in a reduction of the event recovery when comparedto the MHP77.

Maize calli were also screened for integration of the transgene cassettefrom the donor DNA vector (PHP45970, PHP50238, PHP53132, PHP53134,PHP53136, PHP53133, PHP53135, PHP53137, and PHP50239) at the MHP77recognition site through junction PCR and selected callus events wereregenerated into T0 plants. When integration occurred, e.g. the donorsequence was integrated at the recognition site. Insertion (Table 7 and8) is designated as “Yes”. When no integration occurred, Insertion isdesignated as “no”.

Example 12 Creation of MS26 Variant Meganucleases

A. MS26+& MS26++ Meganucleases and MS26 Recognition Site

An endogenous maize genomic target site comprising the MS26 recognitionsite (SEQ ID NO: 269) was selected for design of a custom double-strandbreak inducing agent. The MHP26 recognition site is a 22 bppolynucleotide and having the following sequence: (SEQ ID NO: 269)gatggtgacgtacgtgccctac

Wild type I-CreI meganuclease (SEQ ID NO: 3) was modified to produce twoengineered meganucleases, MHP26+ (SEQ ID NO: 270) and MHP26++ (SEQ IDNO: 271), designed to recognize the MHP26 recognition sequence. Thedesign of custom made meganucleases has been described in United StatesPatent Application Publication No. US 2007/0117128 A1.

B. MS26 Variant Meganucleases

As described in Example 6 and 9, LIG3-4 variants were introduced intoyeast and maize and demonstrated significantly higher meganucleaseactivity when compared to the non-variant LIG3-4 meganuclease. TheseLIG3-4 variants were characterized with specific amino acidmodifications when compared to the parental LIG3-4 (Table 2 and FIG.5A-5E). To test if these amino acid modification ( and respectivenucleotide modifications) can also increase the activity of a MS26+meganuclease, the exact same nucleotide/amino acid modifications asdescribed for LIG3-4 (7), LIG3-4 (15), and Lig3-4(B65) were introducedinto MS26+ through standard molecular biology techniques, resulting inthe following three MS26+ variants: MS26+ (7) (SEQ ID NO: 272), MS26+(15)(SEQ ID NO: 273), and MS26 (B65) (SEQ ID NO: 274) variants.

Similarly, the MS26++ nucleotide/amino acid sequence was optimized toinclude the nucleotide/amino acid modifications of LIG3-4 (15) resultingin MS26++(15) meganuclease variant (SEQ ID NO: 275).

Example 13 Analysis of Meganuclease Activity of MS26+ and MS26++Variants in Maize

Genes encoding the MHP26+ and MHP26++ engineered meganucleases wereoptimized for expression in plants. The engineered meganucleaseexpression cassettes contained the maize codon-optimized nucleotidesequences for better performance in maize cells. The meganuclease genesequences were also supplemented with DNA sequences encoding a SV40nuclear localization signal (SEQ ID NO: 34) resulting in the plantoptimized sequence of SEQ ID NO: 276 for MHP26+ and SEQ ID NO: 279 forMS26++. The maize ubiquitin promoter and the potato proteinase inhibitorII gene terminator sequences completed the endonuclease gene designs.Plant optimized sequences for MS26+ and MS26++ variant meganucleases areSEQ ID NO: 419, 277-279 and SEQ ID NO: 280, respectively.

A. Vector Construction for Plant Expression Vectors of the MeganucleaseGenes and Repair (Donor) DNAs for Transgene Integration by HomologousRecombination

Coding parts of the MS26+ variants were introduced into the test vectorPHP51583 containing a slot for meganuclease driven by ubiquitinpromoter, a fusion of two marker genes, MoPAT and DsRed, also under thecontrol of ubiquitin promoter, and kanamicyn resistance gene.

The resulting constructs were delivered into the scutellum cells ofmaize immature embryos via microprojectile bombardment as described inExample 1. Developmental genes (BBM and WUS) were also delivered byco-bombarded (Example 1 and 2).

B. Meganuclease Activity of MS26+ Variants in Maize

Callus tissue of transgenic events was collected, total genomic DNA wasextracted and used as a template to amplify DNA fragment of about 1 kbcomprising the Ms26 recognition site. Frequencies of mutations of theMS26 recognition site (Target site mutation rate) were estimated by thefragments digestion with BsiWI restriction nuclease which cuts theintended Ms26 recognition site. Frequency of mutations was calculatedbased on the percentage of remaining (uncut) fragment indicatingmutations at the target site. Events with at least 50% of undigestedfragment were indicative of at least one allel being cut in first stagesof development and thus were indicative of mutations. Unlike in the caseof LIG3-4 (Example 9), no decrease in frequency of event recovery of theMS26+ variants was observed when compared to the parental MS26+. Allthree MS26+ variants yielded higher mutation frequencies compared withMs26+ meganuclease (Table 9). While Ms26+ (B65) and Ms26+ (7)demonstrated moderate increase in meganuclease activity (3 and 4 foldincrease, respectively), Ms26+(15) demonstrated approximately a 10 foldincrease of activity (Table 9).

TABLE 9 Activity of MS26+ and MS26+ variant meganucleases as determinedby target site mutation rate (TS mutation rate) in plant tissue. Numberof events TS Mutation Meganuclease analyzed Rate MS26+ 282 2% MS26+ (7)191 9% MS26+ (15) 227 25% MS26+ (B65) 176 7%

Introducing the same amino acid modifications (mutations) as LIG3-4 (15)into MS26++ (15) resulted in a dramatic increase of meganucleaseactivity as measured by the % mutation rate of MS26++ (44%) whencompared to MS26 (7%) (Table 10). This data indicates that nearly halfof all events analyzed carried mutations at the Ms26 recognition site.

TABLE 10 Activity of MS26+ and MS26+ variant meganucleases as determinedby target site mutation rate (TS mutation rate) in plant tissue. Numberof events TS Mutation Meganuclease analyzed Rate MS26++ 189 7% MS26+(15) 185 44%

Example 14 Creation of MHP and MHP14+ Variant Meganucleases

A. MHP14 & MHP14+ Meganucleases and MHP14 Recognition Site

An endogenous maize genomic target site comprising the MHP14 recognitionsite (SEQ ID NO: 281) was selected for design of a custom double-strandbreak inducing agent. The MHP14 recognition site is a 22 bppolynucleotide located and having the following sequence:

(SEQ ID NO: 281) caaacagattcacgtcagattt

Wild type I-CreI meganuclease was modified to produce the engineeredmeganucleases MHP14 (SEQ ID NO: 282) and MHP14+ (SEQ ID NO: 283)designed to recognize the MHP14 recognition sequence. The design ofcustom made meganucleases has been described in United States PatentApplication Publication No. US 2007/0117128 A1.

B. MHP14 and MHP14+ Variant Meganucleases

Variants of the MHP14 meganuclease were created through gene shufflingmethods in a manner similar to how the LIG3-4 variants were created anddescribed in Example 3. This involved the introduction of amino acidmodifications as found in naturally occurring meganuclease proteins andpreviously identified in LIG3-4 variants as well as random mutation. Theshuffling process resulted in generation of MHP14 variants withrecombination of amino acid modifications, unintended amino acidmodifications due to mutagenic PCR, deletions, and insertions (SEQ IDNOs: 284-298) Corresponding DNA sequences are SEQ ID NO: 300-314.

Mutations from five MHP14 variants, MHP14 (04), MHP14 (06), MHP14 (08),MHPP14 (12) and MHP14 (14), were introduced into MHP14+, resulting inMHP14+ (04) (SEQ ID NO: 315), MHP14+ (06) (SEQ ID NO: 316), MHP14+ (08)(SEQ ID NO: 317), MHP14+ (12) (SEQ ID NO: 318), MHP14+ (14) (SEQ ID NO:319), respectively. One additional variant was generated by introductionof the G19S mutation from LIG3-4 (15) into MHP14+, resulting in MHP14+(15) (SEQ ID NO: 320). These mutations were introduced into MHP14+through standard molecular biology techniques.

Example 15 Analysis of Meganuclease Activity of MHP14 and MHP14+Variants in Yeast and Maize

A total of 15 MHPP14 variants with increased activity were confirmed inthe yeast system (as described in Example 6). Increased activity wasobserved across a range of temperatures: 28 degrees Celsius, 34 degreesCelsius and 37 degrees Celsius, as shown in Table 11.

TABLE 11 Activity of MHP14 variant meganucleases in yeast ScreeningStrain assayed at different temperatures. meganuclease 28° C. 34° C. 37°C. MHP14 0 2 2 MHP14(L14-07) 0.5 4 MHP14(01) 0 3 3 MHP14(06) 0 3.5 3.5MHP14(L14-04) 0 3 MHP14(08) 1 4 4 MHP14(07) 0.5 2.5 2.5 MHP14(03) 0.5 33 MHP14(04) 2 x x MHP14(02) 2 4 4 MHP14(13) 1 4 3.5 MHP14(L14-03) 0 3MHP14(14) 1 4 4 MHP14(09) 2 4 4 MHP14(12) 1.5 4 4 MHP14(10) 1 4 4

Large increases in meganucleaseactivity (high scores) were observed.

FIG. 10A-FIG. 10D show the amino acid modifications of MHP14 variantmeganucleases relative to the MHP14 parental meganuclease. A (-)indicates that the amino acid is identical to MHP14.

Results from activity screening of five MHP14+ variants are shown inTable 12.

TABLE 12 Activity of MHP14 + variant meganucleases in yeast ScreeningStrain assayed at different temperatures. Meganuclease 28° C. 37° C.MHP14 0 2 MHP14+ (04) 2 X MHP14+ (06) 0 4 MHP14+ (08) 1 4 MHP14+ (12) 24 MHP14+ (14) 1 4 MHP14+ (15) — —

All MHP14+ variants showed higher activity in the Yeast Assay screenedat 37 C when compared to the MHP14 meganuclease (Table 12). VariantMHP14+ (04), MHP14+ (08), MHP14+ (12) and MHP14+ (14) showed increasedactivity even when assayed at lower temperatures temperature of 28° C.(Table 12.)

Genes encoding the MHP14 and MHP14+ variant meganucleases were optimizedfor expression in plants. The engineered meganuclease expressioncassettes contained the maize codon-optimized nucleotide sequences forbetter performance in maize cells. The meganuclease gene sequences werealso supplemented with DNA sequences encoding a SV40 nuclearlocalization resulting in the plant optimized sequences of SEQ ID NOs:321-327. The maize ubiquitin promoter and the potato proteinaseinhibitor II gene terminator sequences completed the endonuclease genedesigns.

Testing and analysis of meganuclease activity of the MHP14+ variantsin-planta was performed as described for Ms26+ and Ms26++ variants(Example 12) and results are shown in Table 13.

TABLE 13 Activity of MHP14 and MHP14+ variant meganucleases in maize asdetermined by target site mutation rate (TS mutation rate) in planttissue. Number of events TS Mutation Meganuclease analyzed Rate MHP14192 13% MHP14+ (04) 192 38% MHP14+ (06) 192 7% MHP14+ (08) 192 25%MHP14+ (12) 192 47% MHP14+ (14) 192 39% MHP14+ (15) 192 20%

Two variants, MHP14+ (04) and MHP14+ (08), while demonstrating higheractivity also showed rather high levels of toxicity. MHP14+ 06 showed nodifference in both toxicity and activity when compared to MHP14. Twovariants, MHP14+ (12) and MHP14+ (14), demonstrated high levels ofactivity without increased toxicity. MHP14+ (15) variant showed moderateincrease of activity and no increase of toxicity (Table 13).

Example 16 DNA Shuffling to Create Variants of MP107 Meganuclease

An endogenous maize genomic target site comprising the MP107 recognitionsequence (SEQ ID NO: 328) was selected for design of a customdouble-strand break inducing agent. The MP107 recognition site is a 22bp polynucleotide having the following sequence:

ctagtatacgtgagagaccttg (SEQ ID NO: 328).

An engineered MP107 meganuclease (SEQ ID NO: 329) was produced asdescribed in Example 3.

The first phase of MP107 meganuclease optimization was designed tointroduce amino acid modifications into the MP107 meganuclease asdescribed in Example 3. Libraries were based on introduction ofmutations previously identified in LIG3-4, MHP14 and MHP77 variants withincreased activity.

The shuffling process resulted in generation of variants withrecombination of amino acid modifications, unintended amino acidmodifications due to mutagenic PCR, deletions, and insertions (SEQ IDNOs: 330-341). Corresponding nucleotide sequences are shown in SEQ IDNOs:343-354.

A total of 6 MHP107 variants with increased activity were confirmed inthe yeast system (as described in Example 6). Increased activity wasobserved across a range of temperatures: 28 degrees Celsius, 30 degreesCelsius and 37 degrees Celsius, as shown in Table 14.

TABLE 14 Activity of MP107 variant Meganucleases in Yeast ScreeningStrain assayed at different temperatures. meganuclease 28° C. 30° C. 37°C. MHP107 0 0 0 MHP107(D1) 0 0 0 MHP107(D5) 0.5 1.5 MHP107(D3) 0.5 2MHP107(D2) 0 0 MHP107(C6) 0.5 1 3 MHP107(C4) 0 0 MHP107(D4) 0 2MHP107(C5) 0 1 MHP107(C1) 2 3 4 MHP107(C2) 0 0 MHP107(D6) 0 0 0MHP107(C3) 0 0

FIG. 11 show the amino acid modifications of MP107 variants relative tothe MP107 parental meganuclease. A (-) indicates that the amino acid isidentical to MP107.

Example 17 DNA Shuffling to Create Variants of Zm6.3 Meganuclease

An endogenous maize genomic target site comprising the Zm6.3 recognitionsequence (SEQ ID NO: 355) was selected for design of a customdouble-strand break inducing agent. The Zm6.3 recognition site is a 22bp polynucleotide having the following sequence: caggctctcgtaaatgcgcctg(SEQ ID NO: 355).

An engineered Zm6.3 meganuclease (SEQ ID NO: 356)) was produced asdescribed in Example 3.

The first phase of Zm6.3 meganuclease optimization was designed tointroduce amino acid modifications into the Zm6.3 meganuclease asdescribed in Example 3. Libraries were based on introduction ofmutations previously identified in LIG3-4, MHP14 and MHP77 variants withincreased activity.

The shuffling process resulted in generation of variants withrecombination of amino acid modifications, unintended amino acidmodifications due to mutagenic PCR, deletions, and insertions (SEQ IDNOs: 357-371). Corresponding nucleotide sequences are shown in SEQ IDNOs:373-387.

A total of 15 Zm6.3 variants with increased activity were confirmed inthe yeast system (as described in Example 6). Increased activity wasobserved across a range of temperatures: 28 degrees Celsius, 30 degreesCelsius and 37 degrees Celsius, as shown in Table 15.

TABLE 15 Activity of Zm6.3 variant Meganucleases in Yeast ScreeningStrain assayed at different temperatures. meganuclease 28° C. 30° C. 37°C. ZM6.3 0 0.5 2 ZM6.3(4) 1 2 4 ZM6.3(3) 0 0 2 ZM6.3(5) 0.5 1 4ZM6.3(H2) 2 2 4 ZM6.3(H3) 2 2 4 ZM6.3(1) 1 1.5 4 ZM6.3(G4) 2 2.5 4ZM6.3(G1) 4 4 4 ZM6.3(G5) 0 0 2 ZM6.3(G2) 2.5 4 4 ZM6.3(H1) 4 4 4ZM6.3(G6) 4 4 4 ZM6.3(G3) 2 2.5 4 ZM6.3(H6) 4 4 4 ZM6.3(H5) 4 4 4

FIG. 12 shows the amino acid modifications of Zm6.3 variants relative tothe Zm6.3 parental meganuclease. A (-) indicates that the amino acid isidentical to Zm6.3.

Example 18 DNA Shuffling to Create Variants of Zm6.22v2 Meganuclease

An endogenous maize genomic target site comprising the Zm6.22v2recognition sequence (SEQ ID NO: 388) was selected for design of acustom double-strand break inducing agent. The Zm6.22v2 recognition siteis a 22 bp polynucleotide having the following sequence:attgctctctcacatactttta (SEQ ID NO: 388).

An engineered Zm6.22v2 meganuclease (SEQ ID NO: 389). was produced asdescribed in Example 3.

The first phase of Zm6.22v2 meganuclease optimization was designed tointroduce amino acid modifications into the Zm6.22v2 meganuclease asdescribed in Example 3. Libraries were based on introduction ofmutations previously identified in LIG3-4, MHP14 and MHP77 variants withincreased activity.

The shuffling process resulted in generation of variants withrecombination of amino acid modifications, unintended amino acidmodifications due to mutagenic PCR, deletions, and insertions (SEQ IDNOs: 390-403). Corresponding nucleotide sequences are shown in SEQ IDNOs:405-418.

A total of 13 ZM6.22v2 variants with increased activity were confirmedin the yeast system (as described in Example 6). Increased activity wasobserved across a range of temperatures: 28 degrees Celsius, 30 degreesCelsius and 37 degrees Celsius, as shown in Table 16.

TABLE 16 Activity of ZM6.22v2 variant Meganucleases in Yeast ScreeningStrain assayed at different temperatures. meganuclease 28° C. 30° C. 37°C. ZM6.22v2 0 0 1 ZM6.22v2(I2) 1 2 x ZM6.22v2(J5) 0.5 4 ZM6.22v2(J8) 1 3ZM6.22v2(J3) 0.5 3 ZM6.22v2(J4) 0.5 3.5 ZM6.22v2(J7) 0.5 3 ZM6.22v2(I6)0.5 1 2 ZM6.22v2(I4) 0 0 3 ZM6.22v2(I3) 0 0 2 ZM6.22v2(I5) 0 0 0ZM6.22v2(J2) 0 2 ZM6.22v2(I9) 0 2 ZM6.22v2(I7) 0 2 ZM6.22v2(I8) 0.5 2.5ZM6.22v2 0 0 1 ZM6.22v2(I2) 1 2 x ZM6.22v2(J5) 0.5 4 ZM6.22v2(J8) 1 3

FIG. 13 shows the amino acid modifications of Zm6.22v2 variants relativeto the Zm6.22v2 parental meganuclease. A (-) indicates that the aminoacid is identical to Zm6.22v2

Example 19 Use of Different Amino Acid Linkers Sequences to CreateMeganucleases with Increased Activity

As discussed in Example 3, all variant meganucleases comprised a linkerpolypeptide that links the two re-engineered I-CreI monomers into asingle amino chain. The variant meganucleases MHP14(10) (SEQ ID NO: 292)and MHP77(L9-01) (SEQ ID NO: 92) were created as described in Examples.These variant meganucleases were also characterized by having adifferent linker sequence when compared to the linker sequence in theirrespective parent meganucleases (FIG. 15A-FIG. 15D). In MHP14(10), aframeshift occurred at the second codon of the linker E160 and thereading frame was restored at S193, the last residue of the linker. InMHP77(L9-01), a frameshift occurred at the first codon of the linkerW159 and the reading frame was restored at L198. So the first 4 aminoacids of the second unit of the linked dimer were changed. These dataindicates that variant meganucleases can be created with a diverselinker sequence, while still obtaining increased meganuclease activity.

Alignment of the entire amino acid sequence (FIG. 15B) of LIG3-4 (SEQ IDNO: 1), MHP14 (SEQ ID NO: 282) MHP14(10) (SEQ ID NO: 292), MHP77 (SEQ IDNO: 86), and MHP77(L9-01) (SEQ ID NO: 92) revealed a percent identity ofas low as 80.8%. Hence, variant meganucleases were created that hadincreased meganuclease activity while having only 80% similarity to theparental meganuclease.

Example 20 Identification of Amino Acid Modifications in StructuralMotifs of Meganucleases

An analysis of the physical positions of amino acid modificationsresponsible for increased meganuclease activity was performed using athree dimensional structure model of the I-CreI meganuclease dimer(Chevalier B S, Monnat Jr R J, Stoddard B L Nat. Struct. Biol. (2001) 8p.312). Amino acid modifications in alpha helix-1 positions 12, 16 and19 were associated with increased activity observed with severalmeganuclease variants as shown in FIG. 16. Alpha helix-1 encompassesamino acids 8 through 19 on subunit number 1 and amino acids 195 through206 on subunit number 2 in SEQ ID NO: 1. Additionally, amino acidmodifications in alpha helix-5 positions 121, 124, 129, 131 and 132 wereassociated with increased activity observed in several meganucleasevariants as shown in FIG. 16. Alpha helix-5 encompasses amino acids120-135 on subunit number 1 and amino acids 307 through 322 on subunitnumber 2 in SEQ ID NO: 1. We predict that additional amino acidmodifications in alpha helix-1 and alpha helix-5 have the potential toresult in meganuclease variants with increased activity over thecorresponding reference meganucleases.

Example 21 Transfer of at Least One Amino Acid Modification to OtherMeganuclease to Create Variant Meganuclease with Increased Activity

As described in the Example 3-19, any one of the amino acidmodifications identified in Examples 3-19 can be transferred to aparental meganuclease to create a variant meganuclease with increasedactivity. FIG. 14A-FIG. 14F list a subset of variant I-CreI typemeganucleases with increased activity. Anyone of these amino acidmodifications can be combined to create a new variant with increasedactivity.

One embodiment of this invention is the transfer of at least amino acidmodification selected from the group of Y12 to H, G19 to S or A, Q50 toK or R, F54 to I, D56 to L, V105 to A, E124 to R, V129 to A, I132 to Vor T, D153 to M or L, V316 to A or I 319 to V to a parental meganucleasein order to improve the activity of the parental meganuclease.

Example 22 Saturated Mutagenesis to Create Variant Meganucleases withIncreased Activity

Saturated mutagenesis can be performed at any of the amino acidmodification positions described in examples 3-21. Saturated mutagenesiswill result in the production a set of meganucleases wherein one aminoacid position is substituted with one of all possible amino acids. Thisset of meganucleases can then be analyzed for increased activity asdescribed above resulting in identifying more possible modifications foran amino acid position that will result in an increased meganucleaseactivity.

Example 23 Creation and Analyses of TS21 and TS14 Variant Meganucleasesin Soybean

A. TS21 and TS14 Recognition Sites and Meganucleases

An endogenous soybean genomic target site comprising the TS21recognition sequence (SEQ ID NO: 423) or the TS14 recognition sequence(SEQ ID NO: 424) was selected for design of a custom double-strand breakinducing agent. The soybean genomic target sites and design of custommade TS21 and TS14 meganucleases have been described in U.S. patentapplication Ser. No. 13/427,138, filed on Mar. 22, 2012, which isincorporated by reference in its entirety.

B. TS21 and TS14 Variant Meganucleases

To test if the LIG3-4 amino acid modifications (and respectivenucleotide modifications) can also increase the activity of the soybeanTS21 meganuclease and TS14 meganuclease, the exact same nucleotide/aminoacid modifications as described for LIG3-4 (7), LIG3-4 (15), andLig3-4(B65) (Table 1A) were introduced into TS21 meganuclease (SEQ IDNOs: 425 and 429) and TS14 meganuclease (SEQ ID NOs: 433 and 435)through standard molecular biology techniques, resulting in thefollowing three TS21 meganuclease variants and one TS14 meganucleasevariant: TS21(7) (SEQ ID NOs: 426 and 430), TS21(15) (SEQ ID NOs: 427and 431), TS21(B65) (SEQ ID NOs: 428 and 432), and TS14(15) (SEQ ID NOs:434 and 436) variants.

C. Analyses of Meganuclease Activity of TS21 and TS14 Variants inSoybean

Genes encoding the TS21 and TS14 variant meganucleases were optimizedfor expression in plants. The engineered meganuclease expressioncassettes contained the plant codon-optimized nucleotide sequences forbetter performance in soybean. The plant expression vectors for thesesoy variants were made by the same methods as described in U.S. patentapplication Ser. No. 13/427,138. The soybean ubiquitin promoter and thepotato proteinase inhibitor II gene terminator sequences were used forcontrolling meganuclease expression in soybean. The methods used forsoybean transformation, qPCR and genomic PCR assays for the TS21 andTS14 target sites were as described in U.S. patent application Ser. No.13/427,138. The qPCR assays specific to the TS21 and TS14 recognitionsequences were used to identify sequence changes. All hygromycinresistant soybean transgenic events were analyzed by qPCR assays.Changes in the meganuclease target sequence caused by DNA cleavage andrepair result in the copy number reduction of the meganuclease targetsite from two copies in wild type soybean genome to either one or zerocopies in the transgenic events. From qPCR analyses of the TS21 and TS14target sites, it was shown that the copy numbers of the target sites inmost of the positive transgenic events were reduced by half, indicatingone allele of the recognition sites in soybean genome was disrupted bymeganuclease cutting/DNA repair mechanism. As shown in Table 17,introducing the same amino acid modifications (mutations) as LIG3-4variants into the TS21 meganuclease resulted in a dramatic increase ofTS21 target site mutation rates for the TS21(7) variant meganuclease(32.1%) and the TS21(15) variant meganuclease (17.2%), a moderateincrease for the TS21(B65) variant meganuclease when compared to theparental TS21 meganuclease (8.7%). As shown in Table 18, introducing theLIG3-4 (15) mutation into TS14 meganuclease resulted in a decrease ofTS14 target site mutation rate from 16% for the parental TS14meganuclease to 4% mutation rate for the TS14(15) variant meganuclease.

TABLE 17 Activity of TS21 variant meganucleases as determined by targetsite qPCR hit rate (TS mutation rate) in soybean Number of events TSMutation Meganuclease analyzed Rate TS21 184 8.7% TS21 (7) 187 32.1%TS21 (15) 192 17.2% TS21 (B65) 134 12.7%

TABLE 18 Activity of TS14 variant meganuclease as determined by targetsite mutation rate (TS mutation rate) in soybean Number of events TSMutation Meganuclease analyzed Rate TS14 183 16% TS14 (15) 192 4%

What is claimed is:
 1. An isolated or recombinant polynucleotidecomprising a nucleotide sequence encoding a meganuclease polypeptide,said polypeptide comprising: a) an amino acid sequence having at least80% sequence identity to SEQ ID NO:270 and at least one amino acidmodification at an amino acid position corresponding to a position ofSEQ ID NO:270 selected from the group consisting of positions 16, 19,22, 50, 71, 185, 244, 258, 316, and combinations thereof; or, b) anamino acid sequence having at least 1, 2, 3, 4, 5, 6, 7, or 8 of any ofthe amino acid modification of (a).
 2. The isolated or recombinantpolynucleotide of claim 1, wherein said at least one amino acidmodification comprises: a) an isoleucine (I) at a position correspondingto amino acid position 16 in SEQ ID NO:270; b) a cysteine (C) at aposition corresponding to amino acid position 22 in SEQ ID NO:270; c) alysine (K) at a position corresponding to amino acid position 50 in SEQID NO:270; d) a lysine (K) at a position corresponding to amino acidposition 71 in SEQ ID NO:270; e) a glycine (G) at a positioncorresponding to amino acid position 185 in SEQ ID NO:270; f) a glutamicacid (E) at a position corresponding to amino acid position 244 in SEQID NO:270; g) a lysine (K) at a position corresponding to amino acidposition 258 in SEQ ID NO:270; h) an alanine (A) at a positioncorresponding to amino acid position 316 in SEQ ID NO:270; or i) aserine (S) at position 19 in SEQ ID NO:270.
 3. The isolated orrecombinant polynucleotide of claim 1, wherein said nucleotide sequenceencodes a meganuclease polypeptide, wherein said polypeptide furthercomprises: a) an aspartic acid (D) at a position corresponding to aminoacid position 2 in SEQ ID NO:270; b) a histidine (H) at a positioncorresponding to amino acid position 12 in SEQ ID NO:270; c) anisoleucine (I) at a position corresponding to amino acid position 16 inSEQ ID NO:270; d) a serine (S) or an alanine (A) at a positioncorresponding to amino acid position 19 in SEQ ID NO:270; e) a cysteine(C) at a position corresponding to amino acid position 22 in SEQ IDNO:270; f) a leucine (L) at a position corresponding to amino acidposition 23 in SEQ ID NO:270; g) a methionine (M) at a positioncorresponding to amino acid position 24 in SEQ ID NO:270; h) an arginine(R) or an alanine (A) at a position corresponding to amino acid position28 in SEQ ID NO:270; i) an arginine (R), alanine (A), glutamine (Q),cysteine (C), glycine (G), serine (S), threonine (T), leucine (L),glutamic acid (E), or a proline (P) at a position corresponding to aminoacid position 30 in SEQ ID NO:270; j) an arginine (R) at a positioncorresponding to amino acid position 31 in SEQ ID NO:270; k) an arginine(R), alanine (A), lysine (K) glutamine (Q), glycine (G) or a leucine(L)at a position corresponding to amino acid position 32 in SEQ IDNO:270; l) an asparagine (N) at a position corresponding to amino acidposition 36 in SEQ ID NO:270; m) a leucine (L) at a positioncorresponding to amino acid position 43 in SEQ ID NO:270; n) an arginine(R) or lysine (K) at a position corresponding to amino acid position 50in SEQ ID NO:270; o) an isoleucine (I) or a leucine (L) at a positioncorresponding to amino acid position 54 in SEQ ID NO:270; p) a leucine(L) at a position corresponding to amino acid position 56 in SEQ IDNO:270; q) a glutamic acid (E) at a position corresponding to amino acidposition 57 in SEQ ID NO:270; r) an isoleucine (I) at a positioncorresponding to amino acid position 58 in SEQ ID NO:2701; s) ahistidine (H) or alanine (A) at a position corresponding to amino acidposition 59 in SEQ ID NO:270; t) a valine (V) at a positioncorresponding to amino acid position 62 in SEQ ID NO:270; u) a lysine(K) at a position corresponding to amino acid position 71 in SEQ IDNO:270; v) a threonine (T) at a position corresponding to amino acidposition 72 in SEQ ID NO:270; w) an alanine (A) at a positioncorresponding to amino acid position 73 in SEQ ID NO:270; x) a glycine(G) at a position corresponding to amino acid position 79 in SEQ IDNO:270; y) an arginine (R) at a position corresponding to amino acidposition 80 in SEQ ID NO:270; z) a lysine (K) at a positioncorresponding to amino acid position 81 in SEQ ID NO:270; aa) anarginine (R) at a position corresponding to amino acid position 82 inSEQ ID NO:270; bb) an aspartic acid (D) at a position corresponding toamino acid position 86 in SEQ ID NO:270; cc) a leucine (L) at a positioncorresponding to amino acid position 87 in SEQ ID NO:270; dd) anisoleucine (I) at a position corresponding to amino acid position 91 inSEQ ID NO:270; ee) an isoleucine (I) at a position corresponding toamino acid position 95 in SEQ ID NO:270; ff) an arginine (R) at aposition corresponding to amino acid position 98 in SEQ ID NO:270; gg) avaline (V) at a position corresponding to amino acid position 103 in SEQID NO:270; hh) an alanine (A) at a position corresponding to amino acidposition 105 in SEQ ID NO:270; ii) an arginine (R) at a positioncorresponding to amino acid position 111 in SEQ ID NO:270; jj) in aserine (S) at a position corresponding to amino acid position 113 in SEQID NO:270; kk) a proline (P) at a position corresponding to amino acidposition 114 in SEQ ID NO:270; II) an arginine (R) at a positioncorresponding to amino acid position 116 in SEQ ID NO:270; mm) a glycine(G) at a position corresponding to amino acid position 117 in SEQ IDNO:270; nn) a threonine (T) at a position corresponding to amino acidposition 118 in SEQ ID NO:270; oo) a glycine (G) at a positioncorresponding to amino acid position 121 in SEQ ID NO:270; pp) anarginine (R) at a position corresponding to amino acid position 124 inSEQ ID NO:270; qq) a cysteine (C) at a position corresponding to aminoacid position 128 in SEQ ID NO:270; rr) an alanine (A) at a positioncorresponding to amino acid position 129 in SEQ ID NO:270; ss) anarginine (R) at a position corresponding to amino acid position 131 inSEQ ID NO:270; tt) a valine (V) at a position corresponding to aminoacid position 132 in SEQ ID NO:270; uu) a serine (S) at a positioncorresponding to amino acid position 147 in SEQ ID NO:270; vv) analanine (A) at a position corresponding to amino acid position 151 inSEQ ID NO:270; ww) a leucine (L) or a methionine (M) at a positioncorresponding to amino acid position 153 in SEQ ID NO:270; xx) atryptophan (W) at a position corresponding to amino acid position 159 inSEQ ID NO:270; yy) a glutamic acid (E) at a position corresponding toamino acid position 160 in SEQ ID NO:270; zz) a valine (V) at a positioncorresponding to amino acid position 161 in SEQ ID NO:270; aaa) atyrosine (Y) at a position corresponding to amino acid position 162 inSEQ ID NO:270; bbb) an arginine (R) at a position corresponding to aminoacid position 163 in SEQ ID NO:270; ccc) a histidine (H) at a positioncorresponding to amino acid position 164 in SEQ ID NO:270; ddd) aleucine (L) at a position corresponding to amino acid position 165 inSEQ ID NO:270; eee) an arginine (R) at a position corresponding to aminoacid position 166 in SEQ ID NO:270; fff) a histidine (H) at a positioncorresponding to amino acid position 167 in SEQ ID NO:270; ggg) aproline (P) at a position corresponding to amino acid position 168 inSEQ ID NO:270; hhh) an alanine (A) at a position corresponding to aminoacid position 169 in SEQ ID NO:270; iii) a proline (P) at a positioncorresponding to amino acid position 170 in SEQ ID NO:270; jjj) ahistidine (H) at a position corresponding to amino acid position 171 inSEQ ID NO:270; kkk) a proline (P) at a position corresponding to aminoacid position 172 in SEQ ID NO:270; III) an arginine (R) at a positioncorresponding to amino acid position 173 in SEQ ID NO:270; mmm) aleucine (L) at a position corresponding to amino acid position 174 inSEQ ID NO:270; nnn) a proline (P) at a position corresponding to aminoacid position 175 in SEQ ID NO:270; ooo) a glutamine (Q) at a positioncorresponding to amino acid position 176 in SEQ ID NO:270; ppp) analanine (A) at a position corresponding to amino acid position 177 inSEQ ID NO:270; qqq) an arginine (R) at a position corresponding to aminoacid position 178 in SEQ ID NO:270; rrr) a valine (V) at a positioncorresponding to amino acid position 179 in SEQ ID NO:270; sss) aglutamine (Q) at a position corresponding to amino acid position 180 inSEQ ID NO:270; ttt) a valine (V) at a position corresponding to aminoacid position 182 in SEQ ID NO:270; uuu) a proline (P) at a positioncorresponding to amino acid position 183 in SEQ ID NO:270; vvv) a lysine(K) at a position corresponding to amino acid position 184 in SEQ IDNO:270; www) a threonine (T) or a histidine (H) at a positioncorresponding to amino acid position 185 in SEQ ID NO:270; xxx) a serine(S) at a position corresponding to amino acid position 186 in SEQ IDNO:270; yyy) a glutamic acid (E) at a position corresponding to aminoacid position 187 in SEQ ID NO:270; zzz) a leucine (L) at a positioncorresponding to amino acid position 188 in SEQ ID NO:270; aaaa) aglutamic acid (E) at a position corresponding to amino acid position 189in SEQ ID NO:270; bbbb) a glutamine (Q) at a position corresponding toamino acid position 190 in SEQ ID NO:270; cccc) a leucine (L) at aposition corresponding to amino acid position 191 in SEQ ID NO:270;dddd) an amino acid deletion at a position corresponding to amino acidposition 192 in SEQ ID NO:270; eeee) a proline (P) at a positioncorresponding to amino acid position 194 in SEQ ID NO:270; ffff) alysine (K) at a position corresponding to amino acid position 195 in SEQID NO:270; gggg) a serine (S) at a position corresponding to amino acidposition 196 in SEQ ID NO:270; hhhh) a phenylalanine (F) at a positioncorresponding to amino acid position 197 in SEQ ID NO:270; iiii) anisoleucine (I) at a position corresponding to amino acid position 200 inSEQ ID NO:270; jjjj) a valine (V) at a position corresponding to aminoacid position 203 in SEQ ID NO:270; kkkk) a leucine (L) at a positioncorresponding to amino acid position 204 in SEQ ID NO:270; llll) analanine (A) or a serine (S) at a position corresponding to amino acidposition 206 in SEQ ID NO:270; mmmm) a cysteine (C) at a positioncorresponding to amino acid position 209 in SEQ ID NO:270; nnnn) aleucine (L) at a position corresponding to amino acid position 222 inSEQ ID NO:270; oooo) a methionine (M) at a position corresponding toamino acid position 211 in SEQ ID NO:270; pppp) an isoleucine (I) at aposition corresponding to amino acid position 232 in SEQ ID NO:270;qqqq) a serine (S) at a position corresponding to amino acid position236 in SEQ ID NO:270; rrrr) a leucine (L) or an arginine (R) at aposition corresponding to amino acid position 237 in SEQ ID NO:270;ssss) an isoleucine (I) or a leucine (L) at a position corresponding toamino acid position 241 in SEQ ID NO:270; tttt) a glutamic acid (E) at aposition corresponding to amino acid position 244 in SEQ ID NO:270;uuuu) a histidine (H) at a position corresponding to amino acid position246 in SEQ ID NO:270; vvvv) an aspartic acid (D) or histidine (H) at aposition corresponding to amino acid position 253 in SEQ ID NO:270;wwww) an isoleucine (I) at a position corresponding to amino acidposition 254 in SEQ ID NO:270; xxxx) a serine (S) at a positioncorresponding to amino acid position 258 in SEQ ID NO:270; yyyy) anarginine (R) at a position corresponding to amino acid position 267 inSEQ ID NO:270; zzzz) an isoleucine (I) at a position corresponding toamino acid position 278 in SEQ ID NO:270; aaaaa) a tyrosine (Y) at aposition corresponding to amino acid position 281 in SEQ ID NO:270;bbbbb) a phenylalanine (F) at a position corresponding to amino acidposition 282 in SEQ ID NO:270; ccccc) a threonine (T) at a positioncorresponding to amino acid position 289 in SEQ ID NO:270; ddddd) analanine (A) at a position corresponding to amino acid position 292 inSEQ ID NO:270; eeeee) a glycine (G) at a position corresponding to aminoacid position 308 in SEQ ID NO:270; fffff) an arginine (R) at a positioncorresponding to amino acid position 311 in SEQ ID NO:270; ggggg) analanine (A) at a position corresponding to amino acid position 312 inSEQ ID NO:270; hhhhh) an alanine (A) at a position corresponding toamino acid position 316 in SEQ ID NO:270; iiiii) an arginine (R) at aposition corresponding to amino acid position 318 in SEQ ID NO:270;jjjjj) a valine (V) at a position corresponding to amino acid position319 in SEQ ID NO:270; kkkkk) an alanine (A) at a position correspondingto amino acid position 334 in SEQ ID NO:270; lllll) a phenylalanine (F)at a position corresponding to amino acid position 339 in SEQ ID NO:270;mmmmm) a glycine (G) or a leucine (L) at a position corresponding toamino acid position 340 in SEQ ID NO:270; nnnnn) a serine (S) at aposition corresponding to amino acid position 342 in SEQ ID NO:270;ooooo) an asparagine (N) at a position corresponding to amino acidposition 345 in SEQ ID NO:270; ppppp) an asparagine (N) at a positioncorresponding to amino acid position 346 in SEQ ID NO:270; or, qqqqq) anasparagine (N) at a position corresponding to amino acid position 348 inSEQ ID NO:270; or, rrrrr) any combination of a) to qqqqq).
 4. Theisolated or recombinant polynucleotide of claim 1, wherein saidnucleotide sequence encodes a meganuclease polypeptide selected from thegroup consisting of SEQ ID NOs: 272, 273, and
 274. 5. The isolated orrecombinant polynucleotide of claim 1, wherein the polypeptide iscapable of recognizing and cleaving a meganuclease recognition sequenceof SEQ ID NO:269.
 6. The isolated or recombinant polynucleotide of claim1, wherein said polypeptide has an increased meganuclease activity whencompared to a control meganuclease that lacks said amino acidmodification.
 7. The isolated or recombinant polynucleotide of claim 6,wherein said control meganuclease is SEQ ID NO:270.
 8. The isolated orrecombinant polynucleotide of claim 6, wherein the increasedmeganuclease activity is evidenced by: a) a higher yeast assay scorewhen compared to the control meganuclease that lacks said amino acidmodification; or, b) a higher target site mutation rate when compared tothe control meganuclease that lacks said amino acid modification; or, c)a higher in-vitro cutting when compared to the control meganuclease thatlacks said amino acid modification; or, d) any combination of (a), (b)and (c).
 9. The isolated or recombinant polynucleotide of claim 6,wherein the increased meganuclease activity is determined at 16° C., 24°C., 28° C., 30° C. or 37° C.
 10. A recombinant DNA construct, comprisingthe isolated or recombinant polynucleotide of claim
 1. 11. A cellcomprising at least one polynucleotide of claim 1 or the recombinant DNAconstruct of claim 10, wherein said polynucleotide is heterologous tothe cell.
 12. The cell of claim 11, wherein said cell is a plant cell.13. The cell of claim 11, wherein said plant cell is from a monocot. 14.The cell of claim 11, wherein said plant cell is from a dicot.
 15. Aplant comprising a plant cell of claim
 12. 16. A transgenic seedproduced by the plant of claim 15, wherein said transgenic seedcomprises the heterologous polynucleotide of claim 1 or the recombinantconstruct of claim
 10. 17. A method for producing a meganuclease havingincreased activity over a range of temperatures, the method comprising:a) producing a variant meganuclease having at least 80% sequenceidentity to SEQ ID NO:270 by modifying at least one amino acid at anamino acid position corresponding to a position of SEQ ID NO:270selected from the group consisting of positions 16, 19, 22, 50, 71, 185,244, 258, 316, and combinations thereof; b) screening said variantmeganuclease from step a) for the ability to cleave a DNA targetsequence over a range of temperatures between and including 16° C. to37° C.; and c) selecting a variant meganuclease screened in step b) thatis able to cleave a DNA target sequence over said temperature range. 18.The method of claim 17, wherein said range of temperatures comprises: a)16° C.; b) 18° C.; c) 20° C.; d) 24° C.; e) 28° C.; f) 30° C.; g) 37°C.; or, h) any combination of a), b), c), d), e), f), h), g) and g). 19.A method for producing a meganuclease having an increased meganucleaseactivity when compared to a control meganuclease, the method comprising:producing a variant meganuclease having at least 80% sequence identityto SEQ ID NO:270 by modifying at least one amino acid at an amino acidposition corresponding to a position of SEQ ID NO:270 selected from thegroup consisting of positions 16, 19, 22, 50, 71, 185, 244, 258, 316,and combinations thereof; b) screening said variant meganuclease fromstep a) for increased meganuclease activity when compared to a controlmeganuclease; and, c) selecting a variant meganuclease screened in stepb) that is able to cleave a DNA target sequence.
 20. The method of claim19, wherein the increased meganuclease activity is evidenced by: a) ahigher yeast assay score when compared to the control meganuclease thatlacks said amino acid modification; or, b) a higher target site mutationrate when compared to the control meganuclease that lacks said aminoacid modification; or, c) a higher in-vitro cutting when compared to thecontrol meganuclease that lacks said amino acid modification; or, d) anycombination of (a), (b) and (c).
 21. A composition comprising at leastone polynucleotide of claim 1.